Immunophenotyping Profiling in Patients With Primary Nephrotic Syndrome

Primary nephrotic syndrome caused by membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS), or minimal change disease (MCD) is, in each case, associated with dysregulation of specific innate and adaptive immune cell populations. However, the relationship between circulating immune cell subsets and key clinical outcomes, specifically treatment response and disease relapse, remains incompletely defined, and the classical clinical and laboratory markers currently used to monitor these diseases are non-specific and have limited predictive value.

The principal parameter used clinically to monitor treatment response remains 24-hour urinary protein excretion. Complete remission is defined as a reduction in proteinuria to below 0.3 g/24h, and partial remission as a reduction of ≥50% from baseline together with total proteinuria below 3.5 g/24h. Non-response is defined as persistent proteinuria above 3.5 g/24h despite guideline-directed therapy, and relapse as recurrence of proteinuria above 3.5 g/24h in a patient who had previously achieved complete remission.

In MN specifically, serum anti-PLA2R antibody titers provide an additional marker of immunological disease activity: a decline in antibody levels can precede and predict clinical response, while re-emergence or a rise in titer in a patient previously in remission can predict relapse.

Despite the established contribution of specific immune cell populations to primary nephrotic syndrome, the literature remains comparatively sparse regarding abnormalities in regulatory T-cell subsets, monocyte subsets, B-cell subsets (including CD5+ B cells), T-helper subsets, and NK cells, and their potential role in disease pathogenesis across MN, MCD, and FSGS. Furthermore, classical clinical and immunological biomarkers of disease activity and treatment response are non-specific, correlate poorly with the degree of underlying renal injury, and in most cases cannot predict renal outcome. Anti-PLA2R1 antibody titers capture humoral activity and glomerular injury, but only partially explain this heterogeneity, are informative in only 70-80% of patients and frequently lag behind cellular events.

Whether changes in specific circulating immune cell subsets are associated with clinical outcomes in primary nephrotic syndrome, and whether they add predictive value beyond classical markers, remains an open question that this study is designed to address.

This prospective, single-center cohort study will enroll adult patients with either de novo or relapsing primary MN, primary FSGS and MCD, diagnosed by renal biopsy , who underwent rituximab (RTX) or corticosteroids treatment at the University Hospital of Ioannina between JUN 2023 and NOV 2025. Baseline was defined as the time of treatment initiation.

Baseline Assessment - Month 0

• Demographic and clinical data: sex, age, smoking status, alcohol use, obesity, family history, and comorbidities, with particular attention to hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, and coexisting autoimmune disease. A full clinical examination will be performed (systems review, blood pressure, pulse, weight, height, body mass index), and current medications will be recorded.
• Routine laboratory assessment (at baseline and at regular intervals thereafter, per current standards of care for primary nephrotic syndrome): complete blood count, INR, comprehensive metabolic panel (glucose, urea, creatinine, potassium, sodium, calcium, phosphate, AST, ALT, CPK, GGT, alkaline phosphatase, total serum protein, HDL and LDL cholesterol, triglycerides, HbA1c, iron, TIBC, ferritin), inflammatory markers (CRP, ESR), urinalysis, and UPCR/UACR (creatinine, urea, protein, albumin, sodium). Estimated glomerular filtration rate will be calculated using the CKD-EPI creatinine equation.
• Disease-specific serology: in patients with confirmed membranous nephropathy, serum anti-PLA2R antibody titers will be measured per the KDIGO 2021 Clinical Practice Guideline for Glomerular Diseases.
• Annual assessments: viral serology and tumor markers (HBsAg, HBsAb, HBcAb, anti-HCV, AFP, CA 19-9, CA 15-3, CEA, CA-125, PSA); lipid panel (apo-A, apo-B, Lp(a)) and fasting insulin.

After baseline serum creatinine, eGFR (CKD-EPI), albumin, urine protein-to-creatinine ratio (UPCR), urine albumin-to-creatinine ratio (UACR), lipid parameters and inflammatory markers (ESR, CRP) were measured every month after treatment.

Anti-PLA2R antibody levels were measured at baseline and months 3,6 and 12 respectively.

Peripheral-blood immune-cell subsets were analysed by flow cytometry at baseline and at months 3, 6 and 12.

The following immune cell subsets were measured through flow cytometry:

• B-cell compartment: total CD3-CD19+; CD19+CD27+IgD+ (non-switched memory); CD19+CD27+IgD- (class-switched memory); CD19+CD27-IgD+ (naïve mature); CD19+CD5+ (regulatory B cells, Bregs).
• T-cell compartment: CD3+; CD3+CD4+; CD3+CD8+; CD4/CD8 ratio; CD4+CD25+FoxP3+ (regulatory T cells, Tregs); CD4+CD45RA+ (naïve); CD4+CD45RO+ (memory); CD45RA+RO+ (transitional).
• Monocyte compartment: total monocytes; CD14++CD16- (classical); CD14++CD16+ (intermediate); CD14+CD16++ (non-classical); CD14+HLA-DR+ (activated).
• CD16+CD56+ (NK cells).

The study aims to determine whether baseline levels and treatment-related changes in these immune cell subsets are associated with treatment response and disease relapse, and to correlate them with standard clinical and laboratory markers of renal function and disease activity.

Inclusion criteria

• Patients with nephrotic syndrome and a biopsy-confirmed diagnosis of membranous nephropathy, primary focal segmental glomerulosclerosis, or minimal change disease.
• Patients with membranous nephropathy and positive serum anti-PLA2R antibodies, in the absence of histological confirmation by renal biopsy.
• Patients with a previously confirmed diagnosis of primary nephrotic syndrome who present with disease relapse requiring re-induction therapy.

Exclusion criteria

• Patients with nephrotic syndrome in whom membranous nephropathy, primary FSGS, or MCD has not been confirmed by renal biopsy (and who do not meet Inclusion Criterion 2).
• Patients with secondary causes of nephrotic syndrome (malignancy, autoimmune disease, drug-induced, etc.).
• Patients with active malignancy.
• Patients with severe heart failure (NYHA class IV) or hepatic failure.
• Patients with active infection or inflammation.
• Patients receiving, or who received within the preceding 6 months, immunomodulatory agents for an unrelated condition.