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Case report: a 5-year-old with new onset nephrotic syndrome in the setting of COVID-19 infection

• Kelsi M. Morgan   ORCID: orcid.org/0000-0002-5785-0976 1 &
• Peace D. Imani 1 , 2  

BMC Nephrology volume  22 , Article number:  323 ( 2021 ) Cite this article

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This is a case report of an asymptomatic SARS-CoV-2 infection associated with new-onset nephrotic syndrome in a pediatric patient. This is the third case of new-onset nephrotic syndrome in children associated with SARS-CoV-2 infection, but is the first case report describing a new-onset nephrotic syndrome presentation in a patient who had asymptomatic COVID-19 infection.

Case presentation

This is a case of a previously healthy 5 year old female who presented with new-onset nephrotic syndrome in the setting of an asymptomatic COVID-19 infection. She presented with progressive edema, and laboratory findings were significant for proteinuria and hypercholesterolemia. She was treated with albumin, diuretics, and corticosteroid therapy, and achieved clinical remission of her nephrotic syndrome within 3 weeks of treatment. Though she was at risk of hypercoagulability due to her COVID-19 infection and nephrotic syndrome, she was not treated with anticoagulation, and did not develop any thrombotic events.

Conclusions

Our case report indicates that SARS-CoV-2 infection could be a trigger for nephrotic syndrome, even in the absence of overt COVID-19 symptoms.

Peer Review reports

Since the outbreak of the COVID-19 pandemic, more than 20 million people in the United States alone have been infected with the SARS-Cov-2 virus, and over 350,000 of these people have died [ 1 ]. The symptoms and severity of illness vary widely among infected individuals with most impact on the respiratory, cardiovascular, kidney, hematologic, hepatic, cutaneous, gastrointestinal (GI) and nervous systems [ 2 ]. With regard to the pediatric population, as of December 31, 2020, just over 2.1 million cases of Covid-19 had been reported in children with an estimated 0.00–0.08% of all pediatric COVID-19 cases resulted in death [ 3 ]. Children thus seem to be infected with COVID-19 at a lower rate than adults, and tend to have less severe symptoms of disease. Despite this, there have been numerous reports of COVID-19 causing severe disease in children. Presentations of COVID-19 in children vary: the virus can cause severe symptoms upon initial infection, but children are also affected by multisystem inflammatory syndrome in children (MIS-C), which often occurs weeks after initial infection [ 4 , 5 ]. Various presentations of MIS-C have included respiratory failure, myocardial dysfunction, hematological crises, gastrointestinal symptoms, among others [ 6 ].

Relevant to our case report are reported kidney complications especially studies in the adult population. A study by Yang et al. found that 15.4% of 91 adult patients with COVID-19 examined at autopsy had kidney injury at the time of death [ 7 ]. It is believed that kidney injury due to COVID-19 may be secondary to the ability of the virus to bind angiotensin converting enzyme 2 (ACE2) receptors, though the full mechanism may be more complex [ 8 ]. A recent study by Cheng et al. found that almost 44% of hospitalized adult patients had proteinuria, while a smaller number of patients (26.7%) had hematuria [ 9 ]. Proteinuria and collapsing glomerulopathy have also been described in patients with COVID-19 [ 10 ].

In children, kidney dysfunction in patients with COVID-19 has been reported, with AKI one of the most common kidney manifestations, both in absence and presence of MIS-C [ 11 ], [ 12 ]. Although the incidence of proteinuria in children with COVID-19 has not been well defined, there have been two case reports of new onset nephrotic syndrome in the setting of COVID-19 infection [ 13 , 14 ]. In this report, we present another case of new-onset nephrotic syndrome in conjunction with positive COVID-19 testing.

Clinical presentation

A 5-year old, previously healthy female was admitted to the hospital in December 2020 with a 2-week history of periorbital swelling, with progressive involvement of abdominal and ankle swelling. Prior to admission, the child had developed isolated left eye swelling which was not associated with any other symptoms or vision changes. A week later, evaluation at her primary care physician (PCP)‘s office was notable for 4+ protein on urinalysis. The child was then referred to pediatric nephrology for further evaluation and management. In the renal clinic, on examination she was noted to have moderate abdominal swelling with ascites and pretibial edema. She had further gained 2 kg of weight since her visit to the pediatrician’s office 1 week prior. The child’s mother denied any recent illnesses such as fevers, chills, cough, rhinorrhea, congestion, nausea, vomiting, sore throat, gross hematuria, or diarrhea, and there were no known sick contacts. There was no known family history of kidney diseases.

Laboratory findings

Urine analysis revealed 3+ proteinuria with no blood (Table 1 ). Spot urine protein was greater than 2 g/dL and urine protein-to-creatinine ratio greater than 12 mg/mg, which suggested the presence of nephrotic-range proteinuria. The patient had mild hyponatremia with serum sodium of 133 mmol/L. Serum creatinine was 0.27, and BUN was 20. Serum albumin was 2 g/dL, total cholesterol elevated at 307 mg/dL, elevated triglycerides at 644 mg/dL, with normal complements C3 and C4 at 87 mg/dL and 21 mg/dL, respectively. Our patient’s blood work was also notable for elevated D-dimer 2.49 (normal < 0.4 Ug/mL) and partial thromboplastin time (PTT) 39.1 (25.6–32.4) seconds. She had a mildly elevated TSH (5.6 IU/mL) with a normal free T4 (0.9 ng/dL), a low 25-hydroxy vitamin D level, and a low ferritin of 29. Of note, the surveillance COVID-19 testing (RT-PCR, performed via nasopharyngeal swab), which was performed as part of the general hospital admission process during the time of this case report, returned positive for SARS-CoV-2, and further immunoglobulin (Ig) testing was positive for both IgM and IgG antibodies. The SARS-CoV-2 RT-PCR nasopharyngeal swab test was performed using the Hologic Aptima SARS-CoV-2 Assay (approved for emergency use authorization by the US FDA), and the SARS-CoV-2 immunoglobulin testing was performed on the Abbott Architect i1000 platform (approved for emergency use authorization by the FDA).

Clinical course and outcome

The patient received intravenous 25% albumin and furosemide for diuresis, with excellent response. In addition to a fluid and sodium restriction, she started on oral vitamin D supplements and following a negative purified protein derivative (PPD) skin test, she started corticosteroid therapy at 2 mg/kg per day. Throughout her four-day hospital stay, patient remained asymptomatic from COVID-19 perspective. Her vital signs were stable throughout admission (temperature: 36.1–36.9 C, pulse: 79–107 beats per minute, respiratory rate: 14–22 breaths per minute, SpO2: 97–100% on room air). Although our patient was at an increased risk for hypercoagulability from both nephrotic syndrome and COVID-19 infection, we did not initiate anticoagulation therapy. Patient was discharged home after 4 days at only 700 g (0.7 kg) above her baseline weight, as estimated by her recent weight at a PCP visit 2 months prior to admission.

Patient was in complete remission within 3 weeks of starting corticosteroids and urine protein was still negative after 6 weeks of therapy. Her coagulation profile and thyroid studies normalized without intervention; however, she remains positive for both IgM and IgG SARS-Cov-2 antibodies. Despite these positive results, she had still remained asymptomatic from a COVID-19 perspective at her follow up visit.

New-onset nephrotic syndrome following viral illnesses has been reported in literature ( 15 ) and SARS-Cov-2 infection may be one more of these viruses. There are two case reports of new-onset nephrotic syndrome in the setting of COVID-19 disease. We present the third case of new-onset nephrotic syndrome likely triggered by the novel SARS-Cov-2 infection.

In the two published case reports, both children had COVID-19-related symptoms prior to diagnosis of nephrotic syndrome. One of the cases is an eight-year old boy, with the typical age range for idiopathic nephrotic syndrome while the other is a 15-year old boy.

Our patient, however, developed nephrotic syndrome despite being otherwise asymptomatic from a COVID-19 perspective. This suggests that kidney involvement may be possible even in the absence of any other clear COVID-19 clinical symptoms.

An important consideration for our patient was whether or not to treat her with corticosteroids given her COVID-19 positive status, and the potential to worsen her infection. Previous case reports reported giving corticosteroids to patients with COVID-19 and new-onset nephrotic syndrome without negative outcomes, and the decision was thus made to treat our patient with conventional corticosteroid therapy [ 13 , 14 ]. Our patient did well with this course of treatment, and did not have any overt clinical symptoms following initiation of corticosteroid therapy. We were interested to find that our patient continued to test positive for SARS-CoV-2 at her follow up visit 5 weeks after initial diagnosis. While we did not quantify the patient’s antibody titers, which makes it difficult to know whether and at what rate antibodies were declining at this repeat check, studies indicate that SARS-CoV-2 serum IgM begins to decline in the second month after onset of infection [ 16 ]. Thus, the continued presence of serum IgM 5 weeks after an initial positive test may be related to the timing of infection in our patient. At this time, it is not known whether or not steroids influence the rate of antibody decline, though this is perhaps something that could be studied by future researchers. In consonance with childhood nephrotic syndrome we elected not to proceed with kidney biopsy but initiate treatment with corticosteroids first [ 17 ]. Our patient did well, and had normalization of her coagulation profile following treatment of her nephrotic syndrome. She never required anticoagulation.

This is the third case of new-onset nephrotic syndrome in the setting of COVID-19 in children. However, in contrast to previous reports, our patient was asymptomatic with COVID illness. It could be that SARS-Cov-2 virus could be the trigger for new onset nephrotic syndrome. The steroid responsiveness seen in majority of childhood nephrotic syndrome does not seem to be altered by SARS-Cov-2 infection. Need for anticoagulation should be assessed on a case-by-case basis. More studies are needed to understand the impact and long-term outcomes of COVID-19 on new-onset nephrotic syndrome in children. In absence of clinical trials, therapeutic guidelines become more apparent as more cases are reported.

Availability of data and materials

The data used for this case report is available upon reasonable request.

Abbreviations

Angiotensin converting enzyme 2

Acute kidney injury

Coronavirus 19

Gastrointestinal

Immunoglobulin

Multisystem inflammatory syndrome in children

Primary care physician

Purified protein derivative

Partial thromboplastin time

Reverse transcription-polymerase chain reaction

Severe acute respiratory syndrome coronavirus 2

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Acknowledgments

We thank our patient and her family for allowing us to share this case.

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KMM and PDI were involved in the care of the patient. Both KMM and PDI wrote the case report, have read, and approved the paper.

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Morgan, K.M., Imani, P.D. Case report: a 5-year-old with new onset nephrotic syndrome in the setting of COVID-19 infection. BMC Nephrol 22 , 323 (2021). https://doi.org/10.1186/s12882-021-02520-w

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Minimal change disease occurs most often in children, and nephrotic syndrome in adults is more commonly attributed to focal segmental glomerulosclerosis or membranous nephropathy and not often included in the differential diagnosis of adult onset nephrotic syndrome. A Brazilian study explored this topic by looking at the changing incidence of glomerular disease among 2068 adults from 1999–2005. The data obtained found the most common cause of primary glomerular disease to be focal-segmental glomerulosclerosis (29.7%), followed by membranous nephropathy (20.7%), IgA nephropathy (17.8%), and minimal change disease (9.1%) [3] . Similar results were found in a 2006 study out of Minnesota examining the incidence of glomerular disease among 208 patients [4] . It is important for clinicians to include variant presentations of nephrotic syndrome, particularly minimal change disease when evaluating a patient. As the treatment of nephrotic syndrome varies based on the specific etiology, the recognition and diagnosis of MCD is particularly important in the adult population.

Adult cases of minimal change disease, though less common than cases in children, are documented, specifically in patients actively diagnosed with or suspected of having non-Hodgkin lymphoma (NHL). A 2014 retrospective study examined this association in a series of 18 patients with established diagnoses of concurrent MCD and NHL. Researchers reported that, of these patients, 33.3% had Waldenström macroglobulinemia, 27.7% marginal zone B cell lymphoma and 22.2% chronic lymphocytic leukemia. Further investigation into the timing of disease presentation in this population, revealed four patients in whom MCD presented prior to NHL (with an average delay of 15 months), ten patients who were diagnosed simultaneously, and four who were found to have MCD after their NHL presented (average delay 25 months). Regarding management of the two conditions, reappearance of MCD was more likely in patients who had received only steroid therapy versus those who had been given steroids along with chemotherapy (77.8% and 25%, respectively). The authors concluded that MCD is most likely to appear in patients with NHL's of B cell origin, and that the nephrotic syndrome is best managed with a combination of steroids and chemotherapy [5] . These conclusions are relevant to the patient described earlier, as he had been worked up for MCD but demonstrated no signs or symptoms consistent with NHL at the time. Since the latter condition confers a graver prognosis, the patient should have close monitoring for NHL going forward. However, if he were to be diagnosed with cancer and subsequently receive chemotherapy, he could be at increased risk for nephrotic syndrome depending on which medication he receives. A recently-published case described this risk, citing a 57-year-old female being treated with gefitinib for lung adenocarcinoma who was diagnosed with minimal change nephrotic syndrome, prompting her care providers to switch to erlotinib [6] . Awareness of this potential complication and consideration of minimal change nephrotic syndrome as a disease continuum will be also be important components of outpatient care.

Treatment for minimal change disease in adults has largely been accomplished by an approach with non-immunosuppressive in addition to immunosuppressive therapies. Non-immunosuppressive therapy is commonly instituted regardless of the etiology of nephrotic syndrome and typically consists of an angiotensin-converting enzyme inhibitor in order to preserve renal function. Immunosuppressive therapy is much more individualized to the underlying etiology of the nephrotic syndrome. Treatment of minimal change disease primarily employs glucocorticoids. In a study, Nolasco, et al. examined the efficacy of corticosteroid treatment in adult-onset minimal change disease. Researchers analyzed 75 patients who were treated with an initial dose of prednisone of 60 mg/day. 58 patients (81%) achieved complete remission [7] . A similar study out of Japan by Nakayama et al., found 38 out of 62 patients achieved remission within eight weeks of starting glucocorticoid therapy, and another 15 patients after eight weeks [8] . Although glucocorticoid therapy leads to a transient remission in 80–95% of adults with minimal change disease, approximately 50–75% of glucocorticoid-responsive patients will have a relapse at some point [9] . In patients with recurrent relapses, it is recommended to look at additional therapies, including cyclophosphamide, cyclosporine, tacrolimus, or rituximab.

Although uncommon in the adult population, minimal change disease carries a favorable response to glucocorticoid treatment, with 80-90% of patients achieving complete remission. It is important, therefore, for providers to maintain a broad differential when considering the underlying etiology of nephrotic syndrome in adults, as minimal change disease is treatable with a timely diagnosis and early intervention. In addition, it is prudent to look for secondary diagnoses in an adult patient with MCD for hematologic malignancies as they can be present upon initial diagnosis or appear at a later date.

We thank Mohammad N. Saqib, MD, and Michael J. La Rock, MD for assistance in the editing of this case report.

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• Systematic Review
• Published: 20 September 2022

Childhood nephrotic syndrome and the clinical profile of thromboembolism: a systematic review and meta-analysis

• Kayla Dadgar 1   na1 ,
• Yuanxin Xue 2   na1 ,
• Jason Chung 2   na1 ,
• Stephanie Sangar 3 ,
• Mihir Bhatt 4 ,
• Anthony K. C. Chan 4 ,
• Hannah Geddie 5   na2 &
• Rahul Chanchlani 6 , 7 , 8   na2  

Pediatric Research volume  93 ,  pages 1463–1469 ( 2023 ) Cite this article

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Nephrotic syndrome (NS) is a common kidney disease of childhood, affecting 2–7 children per 100,000. A potentially life-threatening complication affecting children with NS is thromboembolism (TE). However, there remains a paucity of information regarding the burden of TE and its associated risk factors in this population. A systematic review was performed on observational studies examining TE events in children with NS, published in Medline, Embase, CINAHL, and CENTRAL, until May 2021. Meta-analyses were separately conducted on the prevalence of TE within articles exclusively studying children with congenital NS and among articles including all forms of NS. Out of 13,626 articles, 22 were included (14,290 children). The pooled prevalence of symptomatic TE among articles including patients with all forms of NS was 3.60% (95% CI 1.95–5.63), which increased to 8.70% (95% CI 5.11–12.96) in articles with exclusively congenital NS patients. Children with steroid-resistant NS were at a higher risk of TE compared to steroid-sensitive children (OR 4.40, 95% CI 1.34–15.59, p  = 0.013). Focal segmental glomerulosclerosis was the most common histology present in patients with TE (51.2%). Children diagnosed with NS have a significant risk of TE, particularly in patients with congenital NS and steroid resistance.

The prevalence of symptomatic thromboembolic (TE) events in children with nephrotic syndrome (NS) was 3.60% (95% CI 1.95–5.63), which increased more than two-fold in children with congenital NS to 8.70% (95% CI 5.11–12.96).

Potential risk factors for TE events in this population include congenital forms of NS and steroid resistance.

This review provides a better estimate of the prevalence of TE in children with NS, while identifying potentially higher-risk populations who may benefit from TE screening and thromboprophylaxis.

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The preparation of this manuscript was supported by funding from the McMaster Medical Student Research Excellence Award.

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These authors contributed equally: Kayla Dadgar, Yuanxin Xue, Jason Chung.

These authors jointly supervised this work: Hannah Geddie, Rahul Chanchlani.

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Department of Medicine, University of Ottawa, Ottawa, ON, Canada

Kayla Dadgar

Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada

Yuanxin Xue & Jason Chung

Department of Health Sciences: Health Science Library, McMaster University, Hamilton, ON, Canada

Stephanie Sangar

Division of Pediatric Hematology/Oncology, McMaster Children’s Hospital, McMaster University, Hamilton, ON, Canada

Mihir Bhatt & Anthony K. C. Chan

Department of Pediatric Endocrinology, McMaster Children’s Hospital, Hamilton, ON, Canada

Hannah Geddie

Division of Nephrology, Department of Pediatrics, McMaster Children’s Hospital, Hamilton, ON, Canada

Rahul Chanchlani

Department of Health Research Methods, Research, and Impact, McMaster University, Hamilton, ON, Canada

ICES McMaster, McMaster University, Hamilton, ON, Canada

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K.D. and H.G. were involved with all aspects of data collection, screening, analysis, and writing of the manuscript. Y.X. and J.C. were involved with the data collection and analyses and the writing and revision of the manuscript. S.S. assisted with the development of the search strategy and data collection. M.B. and A.K.C.C. provided their input on the data analysis and manuscript revision. R.C. supervised all aspects of this project from inception to manuscript preparation.

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Dadgar, K., Xue, Y., Chung, J. et al. Childhood nephrotic syndrome and the clinical profile of thromboembolism: a systematic review and meta-analysis. Pediatr Res 93 , 1463–1469 (2023). https://doi.org/10.1038/s41390-022-02302-6

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Clinical practice guidelines for nephrotic syndrome: consensus is emerging

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Introduction

Nephrotic syndrome is one of the most common chronic kidney diseases (CKD) in children with an estimated incidence of 2.92 (range 1.15–16.9) per 100,000 children per year [ 1 ]. The majority of patients have steroid-sensitive disease (SSNS) that is characterized by complete remission following 4–6 weeks of daily corticosteroid therapy. About 5–15% of patients show lack of complete remission following adequate therapy with corticosteroids and are labeled as steroid-resistant (SRNS). Long-term outcomes are favorable in patients with SSNS, but might not be as satisfactory in patients with SRNS who continue to show nephrotic-range proteinuria.

Based on scientific evidence, advice for treatment put forth by the International Study for Kidney Diseases in Children (ISKDC) [ 2 ] has been modified over the years by scientific societies. While the broad principles of management are similar, there are differences that impact the diagnosis and treatment of the disease. Guidelines from the Indian Society of Pediatric Nephrology (ISPN) that were first published in 2001 were revised in 2008–2009 and more recently in 2021 [ 3 , 4 ]. We discuss these guidelines and compare them to recently published advice from the International Pediatric Nephrology Association (IPNA) [ 5 ], Kidney Disease Improving Global Outcomes (KDIGO) [ 6 ], and the German Society of Pediatric Nephrology (GSPN) (Tables 1 and 2 ) [ 7 ].

Therapy for the first episode

Several randomized controlled trials (RCTs) have investigated the duration and dose of corticosteroid therapy for the initial episode of nephrotic syndrome. Findings from four high-quality multicenter trials on over 800 patients, that compared standard (8–12 weeks) to prolonged (16–26 weeks) initial prednisone therapy, consistently favor limiting initial therapy to 2–3 months [ 8 , 9 , 10 , 11 ]. An updated meta-analysis comparing 3 months to 5 months or longer initial therapy, in studies at low risk of bias, suggest similar risk of relapse (relative risk, RR, 0.88; 95% CI 0.70–1.11; 377 participants, 3 studies; I 2  = 53%) or of frequent relapses (RR 0.99; 95% CI 0.74–1.33; 376 participants, 3 studies; I 2  = 35%) [ 12 ]. Likewise, therapy for 2 months versus 3 months or longer was not associated with increased risk of relapse (RR 0.91; 95% CI 0.78–1.06; 637 participants; 5 studies; I 2  = 47%) or frequent relapses (RR 0.99; 95% CI 0.82–1.19; 585 participants, 4 studies; I 2  = 0%). Individual patient data analyses of two European studies suggest that initial therapy for 12 weeks was better than 8 weeks in terms of delaying the time to first relapse (63 vs . 29 days; log rank P  = 0.04) and fewer relapses on short-term follow-up (1.2 vs. 0.8 relapses per year; relative relapse rate 0.66; 95% CI 0.49–0.90) [ 13 ]. Post hoc analyses of the Indian [ 9 ] and British [ 11 ] placebo-controlled RCTs indicated that prolonged therapy in young children might reduce the risk of first relapse, but not frequent relapses [ 9 ]. Similar findings were reported in a recent RCT on patients with onset of disease below 4 years old, which showed that extending the duration of initial therapy beyond 12 weeks postponed the time to first relapse without altering the risk of frequent relapses [ 14 ].

Based on the results of these trials, ISPN recommends that initial therapy should comprise of 6 weeks each of daily and alternate day prednisone therapy (Table 1 ) [ 3 ]. While similar advice was given by the GSPN [ 7 ], KDIGO guidelines recommend that physicians could either follow 8 weeks or 12 weeks initial treatment, with empiric advice to prefer the former in patients with rapid (< 7 days) remission or comorbidities such as obesity or hypertension, and consider prolonged (16–24 weeks) therapy in young patients with delayed remission [ 6 ].

Prednisone is dosed either by body surface area or by weight. While dosing using body weight is convenient, it might result in considerable underdosing, especially in younger children [ 15 ]. However, the evidence that short- and medium-term outcomes are better following dosing of initial therapy based on body surface area versus body weight is equivocal [ 12 , 15 ]. While the ISKDC and GSPN recommend that prednisone be dosed on body surface area, guidelines from ISPN and KDIGO recommend either approach, with preference to surface area-based dosing in young children [ 3 , 6 , 7 ]. Once-daily dosing of prednisone is more convenient than divided doses and is associated with similar time to remission [ 16 ].

Therapy of relapses

The choice of prednisone dose and duration of therapy for relapses has been guided more by convention than evidence. Therapy for relapses has comprised daily prednisone until remission, and then on alternate days for the next 4 weeks, followed by cessation [ 2 , 3 ] or tapering over 4–8 weeks [ 17 ]. The risk of relapse is similar for dosing by weight versus surface area (2 studies, 146 participants; RR 1.03; 95% CI 0.71–1.49) [ 12 ]. Data from two RCTs suggest that the duration of alternate day treatment may be shortened without affecting the time to next relapse or frequency of later relapses. The PROPINE multicenter trial, that compared alternate day prednisone for 5 weeks to the same cumulative dose over 10 weeks, showed similar relapses at 6-month follow-up, suggesting no benefit of prolonged tapering schedules [ 18 ]. Another trial showed that reduced length of alternate-day prednisone, from 4 to 2 weeks, was associated with similar risk of frequent relapses at 12-month follow-up (HR 1.03; 95% CI 0.83–1.23), while allowing significantly reduced steroid exposure [ 19 ]. Further, recent studies have not found a difference in outcomes when comparing conventional to reduced doses of prednisone given daily or on alternate days [ 20 ]. A meta-analysis showed that prednisone dosing at 1 mg/kg/day was associated with similar time to remission as 2 mg/kg/day (2 studies, 79 participants; mean difference 0.71 days; 95% CI − 0.43 to 1.86) and with reduced steroid exposure (mean difference − 20.6, 95% CI − 25.7 to − 15.6 mg/kg) [ 12 ].

If these findings are confirmed in the Dutch (RESTERN, NTR5670) and Indian (CTRI/22/03/041221) trials, there might be potential to reduce the dose or duration of corticosteroid therapy for relapse. Until then, KDIGO, GSPN, and ISPN recommend managing relapses conventionally, with daily therapy with prednisone at 60 mg/m 2 or 2 mg/kg until remission, followed by 40 mg/m 2 or 1.5 mg/kg on alternate days for 4 weeks (Table 1 ) [ 3 , 6 , 7 ].

Frequent relapses and steroid dependence

The identification and appropriate management of patients with frequently relapsing and steroid-dependent nephrotic syndrome is important to reduce the risk of morbidity associated with recurrent relapses as well as the side effects of immunosuppressive medications. Variability in the definitions of relapse and frequent relapses (Table 1 ) and practice variations in prednisone use for sub-nephrotic proteinuria impact the prevalence and severity of corticosteroid toxicity. Given the risks of hypertension, raised intraocular pressure, cataract, short stature, obesity, and myopathy associated with even short-term use of high-dose corticosteroids [ 21 ], frequent relapses are being defined more liberally and at fewer relapses than proposed by the ISKDC [ 2 , 3 ]. The management of frequently relapsing or steroid-dependent nephrotic syndrome varies considerably, based on physician and parent preferences and the availability, cost, and toxicity of medications, rather than evidence of relative efficacy and safety based on prospective trials.

Long-term therapy with prednisone

Guidelines from KDIGO and ISPN recommend 9–12 months of therapy with alternate day prednisone at a dose of approximately 0.5 mg/kg [ 3 , 6 ]. Limited data from comparative or single-limb studies indicate that this strategy results in satisfactory response in 43–82.5% patients and reduced frequency of relapses [ 3 ]. However, these patients are at high risk of corticosteroid toxicity. Data from the Kidney Research Network Registry estimated the incidence of steroid-associated toxicity at 293 per 1000 person-years, with every 1 mg/kg per day increase in steroid dose associated with a 2.5-fold increase in the risk of adverse effects, chiefly hypertension, obesity, diabetes, and fractures [ 21 ]. Hence, the GPN guidelines do not suggest the use of long-term alternate day therapy, except in selected patients [ 7 ]. However, guidelines from India recommend alternate day prednisone therapy (~ 0.5 mg/kg) as the initial strategy for patients with frequent relapses while monitoring closely for adverse effects [ 3 ]. KDIGO suggests the use of lower (< 0.5 mg/kg) doses for the long term [ 6 ]. We support the careful use of long-term prednisone on alternate days, with an overall intent of maintaining balance of benefit over harm. We believe that this initial strategy might help postpone the use of additional medications for a disease that might last a decade or longer.

Upper respiratory tract infections are an important trigger for relapses of nephrotic syndrome. In patients receiving alternate-day prednisone, one non-randomized trial and two RCTs suggest that the frequency of relapses is reduced if prednisone is administered daily for 5–7 days at the onset of an upper respiratory infection [ 12 ]. However, these findings were not confirmed in the recent multicenter PREDNOS-2 trial that failed to show a difference in risk of relapses in patients receiving 6-day daily prednisone compared to placebo [ 22 ]. The differences in findings between PREDNOS-2 and prior studies may reflect variations in disease severity, ethnicity, and study quality. Based on these results and the limited use of long-term prednisone to manage frequent relapses in Germany, the GPN does not recommend use of low-dose prednisone during infections [ 7 ]. However, ISPN and KDIGO advise that patients with frequent relapses receiving alternate-day treatment with prednisone should receive daily therapy at a dose of 0.5 mg/kg/day for 5–7 days, during upper respiratory tract infections (Table 1 ). While awaiting a consensus on this approach, the authors support this recommendation. We do not advise this strategy in patients with infrequent relapses and in those receiving additional non-steroidal immunosuppressive medications.

Two prospective studies, from India, on patients with frequent relapses found lower relapse rates in patients receiving low-dose daily (0.2–0.3 mg/kg/day) compared to standard dose alternate-day prednisone (0.5–0.7 mg/kg) for 12 months, without higher risk of steroid toxicity [ 23 , 24 ]. While awaiting results of a trial examining a lower prednisone dose (0.1–0.15 mg/kg) with longer follow-up (CTRI/2019/01/017091), therapy with low-dose daily prednisone may be considered in selected patients with frequent relapses, as was suggested by the KDIGO and Indian guidelines [ 3 , 6 ].

Steroid-sparing medications

All guidelines recommend the use of steroid-sparing agents in patients with frequent relapses or steroid dependence (Table 1 ). However, few randomized studies have compared various interventions for their relative efficacy and safety [ 25 ], and evidence for their use is based on prospective and retrospective single-limb studies. In randomized studies, the risk of relapse was similar for patients receiving levamisole versus cyclophosphamide (2 studies, 97 children; RR 2.14; 95% CI 0.22–20.95), or MMF (one study, 149 patients; RR 1.11; 95% CI 0.86–1.43); lower for cyclosporine compared to cyclophosphamide (2 studies, 95 patients; RR 0.51; 95% CI 0.35–0.74); and similar for MMF and cyclosporine (2 studies, 82 children; RR 1.90, 95% CI 0.66–5.46) [ 25 ]. A Bayesian network analysis (7 reports, 391 patients) showed that therapy with cyclophosphamide was associated with the lowest relapse rates [ 26 ].

Therapy with levamisole or cyclophosphamide appears more effective in patients with frequent relapses than those with steroid dependence [ 27 , 28 ]. Experience from uncontrolled studies indicates that MMF is perhaps more efficacious in young children [ 29 ] and superior to levamisole in steroid dependence [ 30 ]. Therapeutic drug monitoring, to target area under the curve (AUC) of > 50 mg·h/L for mycophenolic acid, is associated with better efficacy [ 7 , 31 ]. Cyclophosphamide appears to be more effective in patients older than 5–7 years, but its prolonged use carries risk of gonadal toxicity, particularly in peripubertal boys [ 28 ]. Calcineurin inhibitors (CNIs), namely, cyclosporine or tacrolimus, maintain sustained remission in the majority of patients and enable significant steroid-sparing in patients who have failed treatment with alkylating agents [ 25 ]. However, since CNIs have a narrow therapeutic index, therapy requires close monitoring of drug levels and for adverse effects, including acute and chronic nephrotoxicity [ 3 , 6 , 7 ].

Following anecdotal reports of benefit in patients with CNI-refractory or CNI-dependent nephrotic syndrome, therapy with rituximab has rapidly emerged as a potent modality to enable sustained remission in patients with steroid-dependent or frequently relapsing disease [ 32 ]. Therapy with 1–4 doses of rituximab in 159 patients with steroid and/or CNI dependence was associated with sustained remission for 5–11 months, enabling withdrawal of additional immunosuppression [ 32 ]. In randomized studies, therapy with rituximab was associated with reduced risk of relapse at 1 year as compared to placebo, prednisone, or CNI (3 studies, 198 patients; RR 0.63, 95% CI 0.42–0.93) [ 25 ]. Chief concerns associated with therapy have been the risks of infusion reactions (4 studies, 252 children; RR 5.8, 95% CI 1.3–25.3) [ 25 ], severe infections [ 32 ], vaccine hyporesponsiveness [ 33 ], and reactivation of viral infections, such as hepatitis B and JC virus [ 34 ]. Recent studies suggest that therapy may lead to variable hypogammaglobulinemia in 35–58% of children, particularly in patients < 8–10 years old, those with preexisting hypogammaglobulinemia, and those receiving multiple rituximab doses or additional immunosuppression [ 35 ].

Choice of medication

In the absence of a strong evidence base, the choice of steroid-sparing therapy is based on patient age, presence of steroid toxicity, adherence, risk of adverse effects, patient preference, and availability of medications [ 3 , 6 , 7 ]. Patients with frequent relapses tend to be younger at disease onset than those with infrequent relapses and often continue to relapse into adolescence and even adulthood [ 36 , 37 ]. The goals of therapy are to reduce the frequency of relapses and minimize complications associated with relapses and repeated prednisone use, while avoiding significant medication-related toxicity. Therefore, it is pragmatic to accept infrequent relapses while on steroid-sparing medications with modest efficacy and reserve the use of more potent but potentially toxic medications for children who continue to relapse frequently and show features of steroid toxicity.

KDIGO guidelines suggest that cyclophosphamide and levamisole be preferred in patients with frequent relapses, and therapy with MMF, rituximab, CNI, and occasionally cyclophosphamide be considered in those with steroid dependence [ 6 ]. However, the use of cyclophosphamide is restricted in many developed countries, including Germany [ 7 ]. Guidelines from the GPN, therefore, limit the choice of steroid-sparing medications to chiefly CNI or MMF [ 7 ]. Subject to availability, levamisole may be considered in patients with frequent relapses but not steroid dependence [ 7 ]. In contrast, ISPN recommends therapy with levamisole or MMF in patients with milder disease. Use of cyclophosphamide or higher doses of MMF is advised in patients with high steroid-threshold relapses, significant steroid toxicity, severe relapses, or failure of therapy with levamisole. Thus, the use of CNI and rituximab is advised in patients who fail the above therapies.

All guidelines provide details on doses and duration of use for immunosuppressive medications, target drug levels where applicable, expected adverse effects, and frequency and type of monitoring [ 3 , 6 , 7 ]. Figure  1 a indicates a practical approach to managing patients with frequent relapses and steroid-dependent nephrotic syndrome.

Management of a steroid-sensitive nephrotic syndrome with frequent relapses and steroid dependence and b steroid-resistant nephrotic syndrome. a In many regions, the initial approach in children with frequent relapses is the prolonged use of prednisone on alternate days. Steroid-sparing agents are necessary in patients who develop steroid-associated adverse effects and those with steroid dependence. While the choice of agents varies, levamisole or mycophenolate mofetil (MMF) are preferred medications for mild disease. Patients with complicated relapses and significant steroid toxicity may benefit from therapy with MMF at higher doses or cyclophosphamide. Patients who relapse despite therapy with two or more steroid-sparing agents, often termed “difficult-to treat,” are considered for therapy with calcineurin inhibitors (CNIs) and rituximab. b Evaluation in steroid resistance focuses on exclusion of secondary and monogenic forms of disease, and histology other than focal segmental glomerulosclerosis and minimal change nephrotic syndrome. Monogenic disease, suspected in patients with initial resistance and infantile onset, familial disease, syndromic or extrarenal features, and non-response to CNI, is managed similar to any proteinuric CKD. Other patients with steroid resistance should receive therapy with a CNI, tapering doses of prednisone, and a blocker of the renin–angiotensin–aldosterone system. Patients with complete or partial response should continue receiving the CNI for 1–2 years, followed by its discontinuation, or switch to MMF or rituximab. The outcome in patients with CNI-refractory disease is not satisfactory

Steroid-resistant nephrotic syndrome

The ISKDC defined non-response following 4 weeks each of daily and intermittent dosing with prednisone; 94% of patients who achieved remission did so within the first 4 weeks [ 2 ]. Recent guidelines from IPNA [ 5 ] and KDIGO [ 6 ] define steroid resistance as lack of complete remission despite 4 weeks daily therapy with prednisone (Table 2 ). The guidelines advise that patients with partial remission at 4 weeks be followed up for an additional 2 weeks, with intensified or similar therapy. During this period, physicians should consider additional measures, including genetic studies and/or kidney biopsy, and therapy with blockers of the renin–angiotensin–aldosterone system (RAAS). Patients showing complete remission following additional therapy are labeled as late steroid responder [ 4 , 5 , 6 ].

Until recently, steroid resistance was defined by ISPN as lack of remission despite 4 weeks daily therapy with prednisone. In the current revision, the duration of daily prednisone therapy that qualifies for the definition of steroid resistance was extended from 4 to 6 weeks (Table 2 ) [ 4 ]. An important reason was that 4–6% of patients who do not respond by 4 weeks might do so by 6 weeks [ 2 ]. There is no data to suggest that patients who respond between 4 and 6 weeks show a more difficult subsequent course of illness. The second reason was to synchronize this definition with the 6-week duration of daily therapy for the initial episode [ 3 , 6 ]. Finally, while the diagnosis of complete remission is relatively unequivocal, that of partial remission requires biochemical assessment of urinary protein and the creatinine ratio that might be difficult to uniformly implement across the world.

Similar definitions have been used when defining steroid resistance at onset of illness (initial or primary resistance) and following non-response in a subsequent disease relapse (late or secondary resistance). Available data suggests that this distinction has relevance, since these patients might show differences in pathophysiology, histology, disease progression, and risk of post-transplant recurrence [ 4 , 5 ].

Genetic studies

Approximately 25–33% patients with sporadic initial resistance show significant variations in genes encoding structural and regulatory proteins of the glomerular filtration barrier [ 38 ]. While there are limited studies in Asian patients, these variations are perhaps less common, approaching ~ 15% [ 39 ]. Advances in exome sequencing and bioinformatics and standardization of reporting have enabled rapid and inexpensive screening for these variations. Identifying a monogenic cause has implications for management and prognosis, since these patients are at high risk of non-response to immunosuppression (odds 3.8; 95% CI 2.3–6.5) as well as kidney failure (odds 3.1; 95% CI 2.3–4.1), compared to patients with non-genetic illness [ 38 ]. Guidelines from IPNA and KDIGO emphasize that all patients with initial steroid-resistant nephrotic syndrome should undergo genetic studies [ 5 , 6 ]. Since the occurrence of complete remission following therapy with CNI correlates well with a non-genetic cause, ISPN has preferred a focused approach limiting genetic studies to patients with non-response to CNI, onset of disease during infancy, family history of steroid-resistant illness, syndromic features, or before kidney transplant [ 4 ]. In developing countries, there is a lack of uniform access to genetic testing and expertise in interpretation of variants, and it was felt that a slightly nuanced approach focused on an enriched cohort might be preferable.

Kidney biopsy

Kidney biopsy is an essential investigation for patients with steroid-resistant nephrotic syndrome. All guidelines recommend that patients with steroid resistance should undergo a biopsy to delineate the histology [ 4 , 5 , 6 ]. Almost similar proportions of patients may show focal segmental glomerulosclerosis (FSGS) and minimal change disease [ 40 ]. Few patients may show renal histology suggestive of C3 glomerulopathy, membranous nephropathy, and IgA nephropathy that will require specific management. Apart from identifying an alternative histopathology, the biopsy helps define the extent of tubulointerstitial fibrosis and pattern of glomerulosclerosis that may influence prognosis. Given reports of significant variations in COL4A genes in patients with steroid resistance, electron microscopy is necessary [ 4 , 5 , 39 ]. ISPN advises kidney biopsy in all patients with steroid resistance, except where histological diagnosis is considered unnecessary, e.g., in patients with known or strongly suspected monogenic disease, such as congenital nephrotic syndrome and familial steroid resistance [ 4 ]. IPNA guidelines advise similarly, with preference for genetic testing before kidney biopsy, provided the results are likely to be available within a few weeks. Given limitations in access to genetic testing and variant interpretation, this advice might not be widely implemented, and kidney biopsy is an important tool for diagnostic evaluation.

The management of patients with steroid resistance is challenging because of variable response to immunosuppression, therapy- and disease-related adverse effects, risk of progression to kidney failure, and likelihood of post-transplant recurrence [ 41 ]. With any immunosuppressive strategy, the intent is to induce remission of proteinuria, which is associated with favorable long-term outcomes [ 42 ].

Therapy with either cyclosporine or tacrolimus was shown to induce remission in 70% (95% CI 64.1–75.3%) of 250 patients with steroid resistance enrolled in 9 randomized trials [ 43 ]. The use of either CNI was associated with higher rates of remission at 6–12-month follow-up than no treatment (2 RCTs; RR 3.5; 95% CI 1.0–9.6), or therapy with IV cyclophosphamide (3 RCTs; RR 1.98; 95% CI 1.25–3.13) [ 44 ]. Further, the two medications had comparable efficacy (2 RCTs; RR 1.1; 95% CI 0.9–1.3) and similar risk of nephrotoxicity [ 45 ]. Prolonged CNI therapy is associated with irreversible chronic nephrotoxicity, observed in 10–25% of patients treated for > 2 years, particularly in the presence of persistent heavy proteinuria, hypertension, and use of high CNI doses [ 46 ].

Based on evidence of efficacy of various medications in inducing and maintaining remission [ 44 ], the KDIGO, IPNA, and ISPN guidelines propose that the standard of care for steroid-resistant nephrotic syndrome should be a combination of CNI, prednisone on alternate days, and angiotensin converting enzyme inhibitor (ACEI) [ 4 , 5 , 6 ]. Tacrolimus is preferred to cyclosporine due to lack of cosmetic adverse effects. Cyclosporine is preferred in patients with seizures or at risk of diabetes. Given the unsatisfactory response to therapy with IV cyclophosphamide, the use of this medication is advised only in patients with contraindication to CNI and in regions where CNI is not available [ 4 , 5 , 6 ]. The use of oral cyclophosphamide or pulse corticosteroids for induction of remission is not recommended.

Therapy with CNI requires dose titration in view of variable bioavailability, with risk of non-response and toxicity. Trough levels for both medications are defined and consistent across guidelines. Most patients remit by median of 2–4 months [ 44 ]. Those showing non-response (lack of complete or partial remission) despite 6 months of therapy are considered CNI-refractory and should be counseled regarding outcomes, screened for monogenic defects if not tested earlier, and considered for other immunosuppressive medications or discontinuation of CNI [ 4 , 5 , 6 ].

In the absence of studies to determine the appropriate duration of CNI therapy in patients with complete or partial remission, KDIGO and IPNA recommend therapy for at least 1 year, while ISPN advises therapy for 2 years or longer [ 4 , 5 , 6 ]. Therapy should be monitored closely for development of adverse effects. Discontinuing CNI may be attempted in patients in sustained remission at 2–3 years of therapy [ 4 , 5 , 6 , 43 ]. Patients who require continued CNI therapy for longer than 2–3 years in view of disease relapses should be managed on its lowest effective dose and considered for repeat kidney biopsy to detect nephrotoxicity [ 11 , 12 ]. Some patients with relapsing illness might be switched to alternative therapies, e.g., MMF or rituximab. The precise time at which the switch should occur is not clear. One randomized trial was terminated prematurely due to low rates of remission in patients switched to MMF after 6 months of therapy with tacrolimus, compared to those who continued with tacrolimus (44.8% vs. 90.3%) [ 47 ].

CNI-refractory steroid-resistant nephrotic syndrome

Unlike the KDIGO and IPNA guidelines, ISPN does not recommend a switch to MMF monotherapy in patients with CNI-refractory disease [ 4 , 5 , 6 ]. However, a combination of CNI and MMF was shown to induce remission in 67.3% of 49 patients included in four case series of patients with CNI-refractory illness, without significant adverse events [ 31 ]. This combination can, therefore, be considered in patients with CNI-refractory disease.

The other medication suggested for patients with CNI-refractory disease is rituximab. Of 234 patients with CNI-refractory disease treated with rituximab, in 13 case reports and 10 case series, complete or partial remission was observed in 50.4% (range 18.8–80%) [ 48 ]. The response rates were higher for patients with minimal change versus FSGS and for late versus initial resistance [ 32 , 48 ]. All recent guidelines therefore recommend rituximab as an option for patients with CNI-refractory disease.

Adjunctive therapies

Therapy with prednisone on alternate days and a RAAS blocker are administered together with the above immunosuppressive medications. Guidelines recommend that the dose of prednisone be tapered from 30 to 40 mg/m 2 (1–1.5 mg/kg) on alternate days and discontinued by 6–12 months [ 4 , 5 , 6 ]. Since disease relapses may now be steroid sensitive, they are treated with daily and alternate day prednisone as in steroid-sensitive illness, apart from ensuring adequate therapy with CNI [ 4 , 5 , 6 ].

All guidelines recommend concomitant use of an ACEI or aldosterone receptor blocker (ARB) in all patients with steroid resistance in order to reduce proteinuria, which is linked to progression of CKD [ 4 , 5 , 6 ]. While dual blockade with ACE and ARB may further reduce proteinuria, their concomitant use is not recommended due to the high risk of adverse effects [ 4 , 5 , 6 ]. Therapy with RAAS blockers should be withheld in the presence of hypovolemia and/or acute kidney injury (AKI). While sparsentan, a dual-acting ARB and highly selective endothelin type A receptor antagonist, was shown to reduce proteinuria more effectively than irbesartan [ 49 ], given its limited availability, the agent is not yet recommended for patients with steroid resistance.

Supportive care and management of complications

Principles of management of complications and components of supportive care are similar across guidelines by IPNA, KDIGO, GSPN, and ISPN [ 3 , 4 , 5 , 6 , 7 ]. Anasarca, hypovolemia, AKI, and serious infections are the chief serious morbidities, especially in patients with steroid-resistant nephrotic syndrome. Persistent proteinuria predisposes to thrombosis, undernutrition, dyslipidemia, and subclinical hypothyroidism or hypovitaminosis D [ 4 , 5 , 6 ]. All guidelines provide fairly similar advice on the management of these complications and outline the principles of immunization and transition of care [ 4 , 5 , 6 ].

Patients with monogenic disease, persistent non-response, recurrent AKI, and severe tubulointerstitial chronicity (e.g., following prolonged CNI use) may progress to advanced CKD, the complications of which are well known. Guidelines from IPNA and ISPN provide similar advice on management in preparation for and following transplantation, including management of post-transplant disease recurrence [ 4 , 5 , 6 ].

Conclusions

Experience over the last 5 decades has shown that the clinical profile, pathology, and outcomes of idiopathic nephrotic syndrome are similar worldwide. Well-planned prospective studies have helped refine treatment protocols for patients with steroid-sensitive and steroid-resistant disease. Thus, there is evidence-based consensus on the dose and duration of initial prednisone therapy for steroid-sensitive disease and choice of calcineurin inhibitors for management of patients with steroid resistance. There is still a need for multicenter randomized controlled studies on management of frequent relapses and steroid dependence, especially in relation to rational use of corticosteroids and steroid-sparing strategies, and on switching therapy from CNI to alternative agents in patients with steroid resistance. Guidelines from the ISKDC, KDIGO, and more recently from IPNA have been modified and adopted across the world, depending on availability of medications and preference by parents and physicians. Review of country-specific guidelines shows that despite minor differences, there is reasonable consensus on the management of both steroid-sensitive and steroid-resistant nephrotic syndrome in children. Harmonizing guidelines and considering region-specific preferences will be useful for uniform guidance for physicians, parents, and patient groups, as well as allow comparisons of outcomes.

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Sinha, A., Bagga, A. Clinical practice guidelines for nephrotic syndrome: consensus is emerging. Pediatr Nephrol 37 , 2975–2984 (2022). https://doi.org/10.1007/s00467-022-05639-6

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DOI : https://doi.org/10.1007/s00467-022-05639-6

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ORIGINAL RESEARCH article

A novel flow cytometry panel to identify prognostic markers for steroid-sensitive forms of idiopathic nephrotic syndrome in childhood.

• 1 Laboratory of Nephrology, Bambino Gesù Children’s Hospital Scientific Institute for Research and Health Care (IRCCS), Rome, Italy
• 2 Division of Nephrology, Bambino Gesù Children’s Hospital Scientific Institute for Research and Health Care (IRCCS), Rome, Italy
• 3 Research Laboratories, Bambino Gesù Children’s Hospital Scientific Institute for Research and Health Care (IRCCS), Rome, Italy

Introduction: The clinical evolution of steroid-sensitive forms of pediatric idiopathic nephrotic syndrome (INS) is highly heterogeneous following the standard treatment with prednisone. To date, no prognostic marker has been identified to predict the severity of the disease course starting from the first episode.

Methods: In this monocentric prospective cohort study we set up a reproducible and standardized flow cytometry panel using two sample tubes (one for B-cell and one for T-cell subsets) to extensively characterized the lymphocyte repertoire of INS pediatric patients. A total of 44 children with INS at disease onset were enrolled, sampled before and 3 months after standard induction therapy with prednisone and followed for 12 months to correctly classify their disease based on relapses. Age-matched controls with non immune-mediated renal diseases or with urological disorders were also enrolled. Demographical, clinical, laboratory and immunosuppressive treatment data were registered.

Results: We found that children with INS at disease onset had significantly higher circulating levels of total CD19 + and specific B-cell subsets (transitional, mature-naïve, plasmablasts/plasmacells, CD19 + CD27 + , unswitched, switched and atypical memory B cells) and reduced circulating levels of Tregs, when compared to age-matched controls. Prednisone therapy restored most B- and T-cell alterations. When patients were subdivided based on disease relapse, relapsing patients had significantly more transitional, CD19 + CD27 + memory and in particular unswitched memory B cells at disease onset, which were predictive of a higher risk of relapse in steroid-sensitive patients by logistic regression analysis, irrespective of age. In accordance, B-cell dysregulations resulted mainly associated with steroid-dependence when patients were stratified in different disease severity forms. Of note, Treg levels were reduced independently from the disease subgroup and were not completely normalized by prednisone treatment.

Conclusion: We have set up a novel, reproducible, disease-specific flow cytometry panel that allows a comprehensive characterization of circulating lymphocytes. We found that, at disease onset, relapsing patients had significantly more transitional, CD19 + CD27 + memory and unswitched memory B cells and those who are at higher risk of relapse had increased circulating levels of unswitched memory B cells, independently of age. This approach can allow prediction of clinical evolution, monitoring of immunosuppression and tailored treatment in different forms of INS.

Introduction

Idiopathic nephrotic syndrome (INS) is the most frequent glomerular disease in childhood, characterized by a damage of the glomerular filtration barrier leading to loss of protein in the urine, hypoalbuminemia and oedema ( 1 ).

At first presentation, patients receive a standard course of oral prednisone, to which the majority of children respond within 4 weeks and are therefore defined as steroid-sensitive (SSNS) ( 1 ). The clinical evolution of SSNS is highly heterogeneous, ranging from non-relapsing or infrequently relapsing forms (NRNS/IRNS), to frequently relapsing forms (FRNS), to patients that are steroid-dependent (SDNS) ( 1 ). Approximately 60% of patients require steroid-sparing immunosuppressive agents and 5-42% of patients continue to experience relapses in adulthood ( 1 ). A minority of patients, 10-15%, are steroid-resistant (SRNS) and at risk of progressing to kidney failure ( 1 , 2 ). Currently, no prognostic markers allow an accurate prediction of clinical evolution at disease onset. Therefore, the initial treatment protocol is standardized for all patients ( 1 ).

For nearly 50 years, INS has been considered a renal manifestation of a systemic T-cell dysregulation ( 3 ). In the past decade, the therapeutic efficacy of anti-CD20 B-cell-depleting monoclonal antibodies, mainly rituximab, has implicated B cells in the pathogenesis of the disease ( 4 , 5 ). In pediatric patients with SSNS, alterations in B-cell homeostasis that can be observed at disease onset, before starting any treatment, have been reported by our group and by others (as reviewed in ( 6 )). To date, several studies have attempted to discriminate SSNS from SRNS forms at disease onset using flow cytometry ( 7 – 9 ). However, comprehensive flow cytometry profiling techniques that allow a complete characterization of lymphocyte subpopulations are not currently available.

To this end, we have set up a reproducible and standardized flow cytometry panel that allows detailed characterization of the lymphocyte subpopulations, in order to define a disease-specific B- and T-cell “signature”. This tool may allow prediction of clinical evolution, treatment monitoring, and tailored therapeutic approaches to different forms of the disease.

Materials and methods

Patient selection.

We conducted a prospective cohort study including all children with INS who presented at disease onset at the Bambino Gesù Children’s Hospital - IRCCS from July 2018 to December 2023. The local institutional review board approved the study, and written informed consent was provided by the participants’ legal guardians/next of kin. The study was performed in compliance with the declaration of Helsinki. Clinical definitions of nephrotic syndrome (IRNS, FRNS, SDNS, SSNS, and SRNS), remission and relapse are listed in Supplementary Table S1 ( 1 ). Inclusion criteria were INS with age at onset below 18 years. Exclusion criteria were congenital or genetic forms of NS, secondary forms of NS, chronic infections, previous treatment with immunosuppressive drugs (excluding low dose steroids for periods <3 months). All patient samples were obtained at disease onset, before starting oral prednisone. The initial therapy was as follows: oral prednisone 60 mg/m 2 /daily for 6 weeks followed by 40 mg/m 2 /every other day for 6 weeks. All patients were followed for 12 months, in order to correctly classify their disease based on relapses. Per internal protocol, renal biopsy was performed only in patients aged ≤1 years or ≥12 years old at disease onset, if clinical finding suggestive of other glomerular disorders were present, or in the absence of response to prednisone therapy after 4 weeks. Age-matched controls with non immune-mediated renal diseases or with urological disorders were also enrolled. Exclusion criteria for control patients included chronic renal failure (estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 m 2 ), chronic infections, previous treatment with immunosuppressive drugs (excluding low dose steroids for periods <3 months). Demographical, clinical, laboratory and immunosuppressive treatment data were registered for all patients during the follow-up.

Laboratory data

Whole blood cell count, serum creatinine, serum albumin, serum total proteins, serum C reactive protein, serum cholesterol and the urine protein-to-creatinine-ratio were recorded. Nasopharyngeal aspirates were obtained if patients presented with respiratory symptoms at onset. CRP > 0.5 mg/dl or positivity at the nasopharyngeal aspirate were considered signs of bacterial or viral infection.

Cell collection

Peripheral blood mononuclear cells (PBMCs) were isolated by Pancoll human (Pan Biotech) density-gradient centrifugation after blood collection in EDTA tubes at disease onset and if available after 3 months. Cells were frozen in bovine serum with 10% DMSO and stored in liquid nitrogen until the flow cytometry analysis.

Flow cytometry analysis

To distinguish lymphocyte subpopulations, thawed PBMCs (1x10 6 cells/sample tube) were stained with fluorochrome-conjugated monoclonal Abs directed against the following surface markers in two different tubes as follows: Tube 1: CD3, CD19, CD21, CD24, CD27, CD38, CD45, CD56, IgD, IgM and IgG (BD Biosciences); Tube 2: CD3, CD4, CD8, CD45RA, CCR7, CD25, CD127, CXCR5, CD38, HLA-DR ( Supplementary Table S2 ). Stained cells were analyzed by a FACS BD LSRFortessa (BD Biosciences) (see Figure 1 for gating strategy). Analyses were performed using the program FlowJo, version 10 (Tree Star, Ashland, OR).

Figure 1 Gating strategy. Live CD45 + lymphocytes were identified based on the FSC/SSC lympho-monocyte and singlet gates. Subsets of gated total CD19 + B cells were identified based on the expression of surface markers as follows: transitional (CD38 high CD24 high ), plasmablasts/plasmacells (CD38 high CD24 - ), mature-naïve (CD21 + CD27 - ) and atypical memory (CD21 - CD27 - ) identified in the “Not transitional-not plasmablasts/plasamcells” population; memory B cells were defined as CD19 + CD27 + cells and memory subclasses were defined as unswitched memory (IgM + IgD + , also known as IgM memory), switched memory (IgM - IgD - ), IgM only memory (IgM + IgD - IgG - ), IgG + switched memory (IgM - IgD - IgG + ) B cells. Subsets of gated total CD3 + T cells were identified based on the expression of surface markers as follows: CD4 + or CD8 + T cells were identified as naïve (CD45RA + CCR7 + ), memory (CD45RA - ), TEMRA (CD45RA + CCR7 - ) and highly activated (CD38 high HLA-DR + ) subsets; CD4 + Tregs (CD25 high CD127 - ) and CD4 + Tfh cells (CXCR5 + HLA-DR - ) were also identified. NK cells were identified as CD56 + cells in CD45 + live gated lymphocytes.

Statistical analyses

Continuous data are expressed as mean ± standard deviation if they passed the normality test (Shapiro–Wilk test), or median and interquartile range otherwise. Categorical data are represented as numbers and percentages. Comparison between INS patients at onset and controls and between relapsing and non-relapsing patients were analyzed by unpaired t test if normally distributed, or nonparametric Mann–Whitney U test. Differences between subgroups were analyzed using a nonparametric Kruskal–Wallis test and, if significant, pairwise comparisons were evaluated by the Dunn’s test. Correlations were tested using the Spearman’s rank order test. The association between several parameters at onset and the risk of relapse during the 12-month follow-up was evaluated by logistic regression model. Covariates were included in multivariable modelling whether they reached a significant p value in univariate analysis. The predictive role of lymphocyte subsets significantly associated with the risk of relapse by multivariable logistic regression was analyzed by receiver operating characteristic (ROC) analysis, and by the Kaplan–Meier method (log-rank test). All p values are two sided and considered statistically significant with p-value <0.05. Analyses were performed using Graphpad Prism 9.0.

Study patients

Overall, 58 pediatric patients were initially enrolled in the study. Fourteen patients were subsequently excluded due to having genetic forms of NS (1 patient), NS secondary to other diseases (2 membranous nephropathy, 1 C3 glomerulopathy), or for having started prednisone before sampling (10 patients) ( Supplementary Figure S1 ). Overall, 44 patients (22 males) were included in the study and 44 age-matched subjects were used as controls (26 males) (13 with congenital anomalies of the kidneys and urinary tract (CAKUT), 8 with kidney cysts, 5 with microhematuria/GBM pathology, 11 with nephrocalcinosis, 7 with a previous history of urinary tract infection). Table 1 and Supplementary Table S3 summarize demographic, clinical and laboratory characteristics at the time of sampling.

Table 1 Patient characteristics.

No significant difference was observed in the demographical characteristics when comparing INS patients to control patients ( Table 1 ). A significantly lower median age at onset was observed in SDNS patients compared to NRNS/IRNS patients ( Supplementary Table S3 ). Estimated (e)GFR and proteinuria were significantly higher in INS patients at onset compared to controls ( Table 1 ). Signs of intercurrent infection were observed in 10 INS patients at disease onset, as determined by CRP > 0.5 mg/dl or positivity at the nasopharyngeal aspirate ( Table 1 ). Signs of infection were not evaluated in control patients, since no nasopharyngeal aspirate was performed in this group. CRP levels were comparable between patients and controls ( Table 1 ). No significant differences were observed in laboratory analyses between subgroups of INS patients, except for lower eGFR in NRNS/IRNS compared to SDNS ( Supplementary Table S3 ). After beginning prednisone therapy, 4 patients did not achieve remission within 4 weeks and were considered SRNS. The median time to achieve remission in SSNS was 9.5 [7.0-14.5] days. At 12 months, 28 SSNS patients relapsed after a median time of 132.0 [88.3-365] days from remission. Overall, 15 patients were classified as NRNS/IRNS, 4 patients as FRNS, 21 patients as SDNS, and 4 patients as SRNS, as defined in Supplementary Table S1 . A second sample was obtained after a mean time of 3.1 ± 0.5 months in 28 INS patients, including 9 patients with NRNS/IRNS, 4 patients with FRNS, 12 patients with SDNS and 3 patients with SRNS: most patients were in complete remission, except for 4 SDNS and 2 SRNS patients who were in active phase of disease. At the time of the second sample collection, 12 patients were receiving immunosuppressive prednisone, which was associated with mycophenolate mofetil or cyclosporin A in 8 patients ( Table 1 ).

Lymphocyte profile

The distribution of the different lymphocyte subsets was evaluated in INS patients at onset and after 3 months of prednisone therapy and compared to age-matched controls ( Table 1 ). At onset, significantly higher median circulating levels of total CD19 + B cells associated with significantly lower median levels of total CD3 + T cells and CD56 + NK cells were found compared to controls ( Table 1 ). Most B-cell subsets (transitional, mature-naïve, plasmablasts/plasmacells, and CD19 + CD27 + , unswitched, switched and atypical memory B cells) were significantly higher in INS patients at onset compared to controls ( Table 1 , Figure 2A ). In contrast, fewer T-cell dysregulations were found in INS patients at onset compared to controls, with a significant reduction of total CD4 + T cells, CD4 + Tregs and CD8 + TEMRA cells and a significant increase of CD8 + naïve T cells and highly activated CD8 + CD38 high HLA-DR + T cells, with no difference in the amount of Tfh cells ( Table 1 , Figure 2B ). No significant correlation was found between signs of infection and each lymphocyte subset, except for a direct correlation with switched memory B cells (r= 0.308, p= 0.042) in INS patients at onset. The completion of the standard course of prednisone therapy normalized or significantly decreased most B- and T-cell alterations ( Table 1 ). In particular, total CD19 + and mature-naïve B cells were strongly and significantly reduced and CD56 + NK cells were significantly increased after 3 months of prednisone therapy compared to both INS at onset and age-matched controls ( Table 1 ). In addition, transitional, plasmablasts/plasmacells, CD19 + CD27 + , unswitched, switched and atypical memory B cells, CD8 + TEMRA and highly activated CD8 + CD38 high HLA-DR + T cells were normalized after prednisone therapy, whilst no significant effect was found on the amount of CD4 + Tregs and CD8 + Naïve T cells ( Table 1 ).

Figure 2 Comprehensive lymphocyte profile representation of idiopathic nephrotic syndrome patients and age-matched controls. tSNE analysis followed by clustering of flow cytometry data defined by the manual gating strategy in Figure 1 . Surface (A) B-cell and (B) T-cell marker distribution in a single idiopathic nephrotic syndrome pediatric patient (INS) and a single age-matched control (CTRL) are shown. tSNE plots were represented as merged (upper panels) or separated (lower panels). The specific identified B-cell and T-cell subsets are indicated by colors. Relative antigen expression was visualized by analysis logarithmic stochastic Heat Map (from blue to red).

To determine whether the lymphocyte profile at disease onset could predict the risk of relapse, the 40 SSNS were subdivided as relapsers and non-relapsers during the 12-month follow-up. No significant difference was found in total CD19 + B cells, CD3 + T cell and CD56 + NK cells ( Supplementary Figure S2 ). Among the different B-cell and T-cell susbets, we found that, at onset, relapsing patients had significantly higher median levels of transitional B cells (3.7% vs 1.9%, p=0.01), CD19 + CD27 + memory B cells (4.3% vs 2.8%, p= 0.01) and of unswitched memory B-cell subsets (2.3% vs 1.4%, p=0.004) compared to non-relapsing patients ( Figure 3 ). In particular, CD19 + CD27 + and unswitched memory B cells were significantly associated with the risk of relapse by logistic regression (OR, 1.9 and 4.6, respectively; p<0.05) and unswitched memory B cells only retained this significant association also when adjusted for age (OR, 3.6; p<0.05). The best threshold for discriminating relapsing from non-relapsing patients identified by ROC analysis was 1.5% of unswitched memory B-cell levels (AUC= 0.79, p= 0.005, Figure 4 ). No further significant difference was found in each other analyzed lymphocyte subset ( Figure 3 , Supplementary Figures S3 , S4 ). In addition, no significant difference was found among relapsers and non-relapsers after 3 months of prednisone therapy ( Figure 3 , Supplementary Figures S2 - S4 ).

Figure 3 B-cell subset profile of steroid-sensitive nephrotic syndrome pediatric patients who experienced or not a relapse event during a 12-month follow-up. Circulating levels of transitional, mature-naive, plasmablasts/plasmacells, atypical memory, total CD19 + CD27 + memory, unswitched memory, switched memory and IgG + switched memory were compared between patients who relapsed or not during a 12-month follow-up as determined at onset (red dots, n=28 vs n=12) or after 3 months of prednisone therapy (blue dots, n=22 vs n=6). Age-matched controls were also represented (green dots, n=40). B-cell subsets were expressed as percentages of total CD45 + lymphocytes. Horizontal lines indicate the medians. Differences between groups were compared using unpaired Mann-Whitney U test.

Figure 4 Unswitched memory B-cell levels at disease onset are predictive of relapse in INS pediatric patients. ROC curve analyzing unswitched memory B-cell levels and the risk of relapse within 12 months of follow-up. The arrow indicates the best cut-off for unswitched memory B cells 1.5%. Relapse-free survival was compared between patients with unswitched memory B-cell levels above (red line) or below (blue line) the cut-off by log-rank test. AUC, area under the curve.

INS patients were subsequently subdivided based on the clinical course of their disease during the follow-up and each subgroup (NRNS/IRNS, FRNS, SDNS, SRNS) was compared with its own age-matched control subgroup ( Supplementary Figures S5 , S6 ). SDNS patients were those with the most relevant B- and T-cell dysregulations ( Supplementary Figures S5 , S6 ). SDNS patients also had significantly higher levels of transitional, mature-naïve and unswitched memory B-cell subsets when compared to NRNS/IRNS, whilst no significant difference was found in any of the analyzed T-cell subsets or in CD56 + NK cells ( Supplementary Table S3 ). Of note, we failed to find a lymphocyte subset discriminating SDNS from FRNS, also due to the low number of patients classified as FRNS.

The aim of this study was to develop a reproducible comprehensive flow cytometry panel to define specific B- and/or T-cell “signatures” in patients with INS. To validate this approach, we have used this technology in children at disease onset, to determine whether specific lymphocyte subpopulations before starting prednisone treatment correlate with median-term prognosis - i.e. with relapse at 12 months - and after 3 months of standard immunosuppressive induction therapy with prednisone to assess the effects of this treatment on lymphocyte profiles. The ultimate goal of this approach is to tailor therapy to individual disease severity and to assess sensitivity to immunosuppressive drugs. Our results show that extended characterization of lymphocyte subpopulations can be performed using only two sample tubes (on for the B-cell and one for the T-cell repertoire). This methodology requires a very limited number of cells, a crucial advantage in a pediatric setting. Antibodies that were used were directed only against surface antigens, as opposed to intracellular epitopes, in order to minimize laborious and less reproducible procedures. We analyzed only cryopreserved cells after thawing not requiring stimulation, which is particularly advantageous as it allows sample shipment to Institutions equipped with the appropriate instruments, facilitating multicentric studies.

Using this methodology, we observed that children with INS at disease onset had higher circulating levels of CD19 + B cells when compared to age-matched controls, before starting immunosuppressive therapies. The sub-analysis of B-cell subpopulations showed significant increase in transitional, mature-naïve, plasmablasts/plasmacells, CD19 + CD27 + , unswitched, switched and atypical memory B cells. When patients were subdivided based on disease relapse during a 12-month follow-up, relapsing patients had significantly more transitional, CD19 + CD27 + memory and unswitched memory B cells at disease onset. Increased unswitched memory B-cell levels were also predictive of a higher risk of relapse in SSNS patients by logistic regression analysis, independently of age. In accordance, B-cell dysregulations resulted mainly associated with SDNS. These results are in agreement with previous studies reporting dysregulations of several sub-population of B-cells in children with INS ( 6 ). In most reports, increased circulating levels of total CD19 + B cells have been observed before therapy, which were reduced after prednisone treatment ( 7 , 8 , 10 – 12 ). Among different B-cell subsets, an expansion of transitional and in particular of memory B cells has been reported more frequently, in both pediatric and adult patients ( 7 , 9 , 11 , 12 ), together with increased mature-naïve B cells in some studies ( 8 , 13 ). A recent characterization of the B-cell transcriptional profile in INS patients in active phase has shown increased expression of genes associated with antibody-secreting B cells, atypical memory B cells, and total memory B cells, both of switched and unswitched “MZ-like” subtype ( 14 ). The novel comprehensive flow cytometry panel described in the current study confirmed these observations and increased the sensitivity of our initial methodological approach which was effective in identifying the expansion of transitional, total memory and switched memory B cells in SSNS patients at disease onset but failed to assess accurately mature-naïve, plasmablasts/plasmacells and unswitched memory B cells, probably due to a different gating strategy ( 11 , 15 ).

In contrast to B-cell dysregulations, we observed modest changes in CD56 + NK cell and T-cell subsets. A significant reduction in Treg cells was also observed, as previously described ( 16 , 17 ).

Stratification of patients in different disease severity classes was limited by the number of patients, which underpowered comparisons. Nonetheless, we observed lower Treg levels compared to control independently from the disease subgroup (i.e. NRNS/IRNS or SDNS), suggesting that lower levels of Tregs are not associated with disease severity, whereas CD8 + CD38 high HLA-DR + T cells were more often increased in SDNS patients. A dysregulation of this highly activated population of T cells has already been reported in SSNS pediatric patients ( 8 ) and its role in the pathogenesis of the disease is under investigation. Expansion of this cell subset can also be observed in hyper-inflammatory conditions such as hemophagocytic lymphohistiocytosis ( 18 ) or acute viral infections ( 19 ).

As expected, prednisone therapy normalized most B- and T-cell subsets, although circulating Tregs were not completely normalized after prednisone therapy, contrary to previous reports, although comparisons are limited by differences in the analyzed time points ( 17 , 20 ). With this methodology, which allows more frequent monitoring without excessive blood amount, we hope in the future to better study the dynamics of Treg levels in pediatric INS.

The goal of this study was primarily methodological. The analysis of patients with INS served primarily as a mean to validate this technology and is therefore limited in its interpretation by the relatively low number of patients, although samples at disease onset are not easy to collect. Nonetheless, rigorous inclusion criteria and a uniform follow-up at 12-months strengthen the analysis and allowed to divide the population in “early” relapsers and patients that had not relapsed at 12 months. With this categorization of patients, differences in B-cell populations at disease onset, even after correcting for patient age, could be observed. Results were also strengthened by the inclusion of an age-matched control population and by repeat analyses after immunosuppressive therapy. Another limitation of this study is that our approach did not allow characterization of Th1/Th2/Th17 dysregulations, which have been demonstrated to sustain active disease state in INS ( 16 , 17 , 21 , 22 ). Currently, the characterization of these Th cell subsets can be performed only by intracellular cytokine staining, which requires more complex sample processing that can compromise the reproducibility of the results ( 23 ), or by the analysis of surface chemokine receptor staining, which is affected by cryopreservation (( 23 ) and our unpublished observations).

In conclusion, we have set up a novel, reproducible, disease-specific flow cytometry panel that allows a comprehensive characterization of circulating B- and T-cells. With this method we found that relapsing patients had significantly more transitional, CD19 + CD27 + memory and unswitched memory B cells at disease onset and identified increased unswitched memory B cell levels as predictors of a higher risk of relapse at 12 months, independently of age. If confirmed, these results may assist in developing different treatment strategies according to the B-cell expression profile, restricting more aggressive immunosuppressive therapy to selected severe patients. They may also provide clues to identify novel and more tailored targets of therapy.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The studies involving humans were approved by Bambino Gesù Children’s Hospital (IRCCS) Ethical Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

Author contributions

MR: Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing, Investigation. FZ: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. AC: Conceptualization, Data curation, Formal analysis, Resources, Validation, Writing – original draft, Writing – review & editing. EC: Conceptualization, Data curation, Methodology, Resources, Writing – original draft, Writing – review & editing. AG: Data curation, Resources, Writing – original draft, Writing – review & editing. MS: Formal analysis, Methodology, Writing – original draft, Writing – review & editing. AR: Data curation, Methodology, Writing – original draft, Writing – review & editing. CB: Data curation, Writing – original draft, Writing – review & editing. FE: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. MV: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. MC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The work was “supported by an unrestricted grant provided by European Society for Paediatric Nephrology”, by the Italian Ministry of Health (5 x 1000 and Ricerca Corrente) and by the European Union - Next Generation EU - NRRP M6C2 – Investment 2.1 Enhancement and strengthening of biomedical research in the NHS. The authors thank the Associazione per la Cura del Bambino Nefropatico ONLUS and Fondazione Bambino Gesù for supporting MV and MC.

Acknowledgments

Author want to thank Dr. Francesco Bellomo for graphical assistance.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1379924/full#supplementary-material

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Keywords: idiopathic nephrotic syndrome, B cell, T cell, lymphocyte profile, prognostic markers

Citation: Riganati M, Zotta F, Candino A, Conversano E, Gargiulo A, Scarsella M, Lo Russo A, Bettini C, Emma F, Vivarelli M and Colucci M (2024) A novel flow cytometry panel to identify prognostic markers for steroid-sensitive forms of idiopathic nephrotic syndrome in childhood. Front. Immunol. 15:1379924. doi: 10.3389/fimmu.2024.1379924

Received: 31 January 2024; Accepted: 14 March 2024; Published: 02 April 2024.

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Copyright © 2024 Riganati, Zotta, Candino, Conversano, Gargiulo, Scarsella, Lo Russo, Bettini, Emma, Vivarelli and Colucci. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Manuela Colucci, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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