INTRODUCTION AND AIM

IgA nephropathy (IgAN) is the most common type of glomerulonephritis worldwide. Its definitive diagnosis requires renal biopsy and the ability to accurately predict individual prognosis is currently limited. Therefore, the identification of new biomarkers allowing non-invasive diagnosis and monitoring of disease activity over time is strongly required. We combined complementary proteomic strategies (SELDI-TOF-MS, two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry (MALDI-TOF-MS/MS)) with the following aims: (i) to determine if analysis of the urinary proteome is helpful in distinguishing patients with biopsy-proven IgAN, (ii) to identify some of the protein(s) that correlate with the severity of histologic lesions, and (iii) to determine if tissue expression correlates with the urinary excretion profile of such proteins.

METHODS

Patients: We first recruited 49 biopsy-proven IgAN patients, 42 patients with non-IgA biopsy-proven chronic nephropathies (CKD: 16 Diabetic Nephropathy, 12 benign Nephroangiosclerosis and 14 Membranous Nephropathy) and 40 healthy controls (CTRL). Next, we enrolled a second group of 14 patients with IgAN and 20 patients with CKD [1 MGN, 7 DN, 6 BNAS, and patients with mesangial proliferative glomerular nephropathy (MPGN) which constitute an independent test set to validation study. Inclusion criteria: age>18 years, absence of nephrotic syndrome, concomitant systemic disease and urological abnormalities and/or infection, serum creatinine levels of ≤2.0mg/dL. IgAN patients were retrospectively classified as progressors (IgAN P) and non-progressors (IgAN NP) on the basis of their clinical outcome at the end of follow-up (51 months, IQR 40.5-65). Patients were considered to have a non progressive course of the disease when they fulfilled both the following criteria: decrease in daily proteinuria >50% of  baseline value (at biopsy) and a stable renal function, as measured by eGFR. All patients were analyzed at the time of histological diagnosis.

SELDI-TOF-MS analysis: Ten μg of urine proteins from each sample were run on CM10 chromatographic surface and the corresponding mass spectra were analyzed on SELDI-TOF/MS (Figure 1). Common mass peaks in the mass range between 3000 and 30000 m/z were identified through ProteinChip DataManager software (BIORAD). The dataset was managed by univariate (clustering) analysis, and a subset of 13 mass peaks was identified which discriminated IgAN patients from both CTRL and CKD (Figure 1). Within this subset, the 2 proteins with the highest m/z were isolated by 2-DE and identified by MALDI-TOF-MS/MS (Figure 2). Finally, proteomic findings were confirmed by immunonephelometry (Figure 3A) and Western Blot analysis of target urine proteins (Figure  3B), whose expression in renal biopsy specimens was explored by immunohistochemistry and confocal microscopy (Figure 4).

RESULTS and CONCLUSIONS

• SELDI-TOF/MS screening of urine samples of recognized a panel of 13 urine peptides which decreased in the urine of in IgAN patients compared to both CKD and healthy subjects (Figure 1);
• Two of the above proteins, were identified as kappa FLC and LG3 (Figure 2)
• Proteomic data were confirmed by IN and WB analyses (Figure 3A and B);
• Renal biopsy analysis by confocal microscopy  revealed an increased deposition of k FLC (Figure 4A) and a decreased expression of LG3 (Figure 4B), which might help explain their pattern of urinary excretion;
• The urine excretion of k FLC and LG3 inversely correlated with the severity of clinical and histological features of IgAN patients (Figure 5).

• The analysis of proteomic data identified LG3 and k FLC as potential non invasive biomarkers to IgAN prognosis.