Monitoring Lupus Nephritis Through Urinary Extracellular Vesicles

Introduction: Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) that can lead to irreversible kidney damage if not detected and managed promptly. LN is classified based on its histopathological features. The classification helps in determining the severity and appropriate treatment strategies for the condition and to rule out other conditions that may mimic the clinical picture of LN. The classification system most commonly used is the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification, which divides lupus nephritis into six classes: I) Minimal Change, II) Mesangial Proliferative LN, III) Focal Proliferative LN, IV) Diffus proliferative LN, V) Membranous LN, VI) Advanced Sclerosing LN. The classification requires invasive kidney biopsies, which are hardly accessible and associated with potential complications and patient discomfort. Furthermore the kidney biopsy only represent a small part of the kidney. Recent research has suggested urinary extracellular vesicles (uEVs) as potential non-invasive biomarkers for kidney diseases, including LN. uEVS are small nano-sized extracellular vesicles of endosomal origin, which are secreted into the urine through fusion of multivesicular bodies with the plasma membrane. Thus, they could represent a promising liquid biopsy that reflects the pattern and/or severity of renal injury.

Hypothesis: The primary objective of this prospective study is to investigate the utility of urinary exosomes as non-invasive biomarkers for monitoring the activity and progression of lupus nephritis.

Methods: SLE patients, fulfilling the 2019 EULAR/ACR classification criteria, referred to kidney biopsy, will be recruited from Department of Rheumatology and Department of Nephrology at Odense University Hospital. Detailed demographic and clinical data including age, sex, ethnicity, medicine, duration of SLE activity, SLE damage and laboratory results will be collected from each patient. Activity of SLE at the time of biopsy will be evaluated using SLE disease activity index 2000 (SLEDAI-2K). SLE damage will be evaluated in accordance with the SLICC damage index. Spot-urine samples will be collected, added protease-inhibitors and frozen at -80 degree. UEVs will be isolated using polyethylene glycol (PEG) precipitation, the concentration of uEV will be determined and protein accessed using western blotting and/or PCR. Glomerular specific uEVs will be isolated and previous developed tests will be applied to detect the membrane attack complex (MAC)/C5b-9 complex. uEVs from plasma/serum will be isolated to test for correlation with uEVs. Kidney biopsies will be histological analyzed at Department of Pathology by experienced nephropathologists according to existing clinical guidelines. Differences in uEV cargo between SLE patients with and without lupus nephritis will be analyzed using appropriate statistical tests. Correlations between uEV biomarkers, histological differences on the kidney biopsy and disease activity will be evaluated using Pearson's correlation analysis.

Perspective: This prospective study aims to establish urinary extracellular vesicles as potential non-invasive biomarkers for monitoring lupus nephritis in SLE patients. The findings from this research may lead to the development of more efficient and patient-friendly approaches for LN management, enabling timely interventions and improved renal outcomes in SLE patients.

SLE patients with kidney involvement

Inclusion criteria

• > 18 years old
• Fulfilling the 2019 EULAR/ACR classification criteria
• Positive autoantibodies, medical history and obejctive examination compatible with SLE
• Referred to kidney biopsy

Exclusion criteria

• Lack of ability or willingness to provide informed consent
• Significant comorbidity, which is considered to potentially impact the outcome

SLE patients with no sign of kidney disease

Inclusion criteria

• > 18 years old
• Fulfilling the 2019 EULAR/ACR classification criteria
• Positive autoantibodies, medical history and obejctive examination compatible with SLE
• Normal plasma creatinine
• Urine albumine/creatinine < 100 mg/g

Exclusion criteria

• Lack of ability or willingness to provide informed consent
• Significant comorbidity, which is considered to potentially impact the outcome

Healthy controls:

Inclusion criteria

• > 18 years old
• No known kidney disease

Exclusion criteria

• Lack of ability or willingness to provide informed consent
• Urine albumin-creatinine ratio > 100 mg/g or proteinuria > 100 mg/day
• Postive autoantibodies
• Significant comorbidity, which is considered to potentially impact the outcome

Biopsy control:

Inclusion criteria

• > 18 years old
• Negative autoantibiodies and immunoglobulines
• Referred to kidney biopsy

Exclusion criteria

• Lack of ability or willingness to provide informed consent
• Significant comorbidity, which is considered to potentially impact the outcome