#4049 nefecon® treatment likely modulates downstream pathways of kidney inflammation and fibrosis in iga nephropathy

Abstract Background and Aims The lifetime risk of kidney failure in patients with IgA nephropathy (IgAN) is substantial. Since the original description of the disease, over 50 years ago, there has been little progress in how we manage this important cause of kidney failure in young adults. KDIGO 2021 Clinical Practice Guidelines recommend goal-directed supportive care as the treatment with the strongest evidence base for the management of all patients with IgAN. The NEFIGAN trial (NCT01738035) tested the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver budesonide to the gut-associated lymphoid tissue-rich distal ileum in patients with IgAN in addition to optimised supportive care. The trial comprised a 6-month run-in, 9-month treatment, and a 3-month follow-up phase. Forty-eight patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day, and 50 patients received placebo. The headline result of the study was that Nefecon® 16 mg/day, added to optimised RAS blockade, reduced proteinuria and stabilised eGFR in patients with IgAN. These findings have now been replicated in the NefIgArd study, which reported in 2021 and provided the basis for the recent FDA and EMA approval of Nefecon® as a treatment for patients with IgAN at high risk of progressive disease. In this study, we determined the composition of urinary proteins from patients treated with placebo and 16 mg of Nefecon® in the NEFIGAN trial using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Method Urine samples from 18 patients from each of the placebo and 16-mg arms of the NEFIGAN trial collected at the start of treatment (SOT) and end of treatment (EOT) were analysed. Patients were only included if they had received at least 8 months of treatment and their urine sample was taken up to 2 days after the completion of tapering. Urine samples were treated to remove abundant albumin and immunoglobulins. The remaining proteins were desalted and then precipitated with 5% trichloroacetic acid and washed twice with ice-cold acetone. The air-dried pellets were dissolved in 45 µL of 100 mM triethylammonium bicarbonate buffer, reduced with 2 mM TCEP (tris(2-carboxyethyl)phosphine), and alkylated with 15 mM chloroacetamide. 500 ng of Sequencing Grade Trypsin was added for enzymatic digestion, carried out overnight at 37°C. The samples were dried to completeness and re-solubilised in 10 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid). LC-MS/MS analysis was performed on an Q Exactive mass spectrometer equipped with a Digital PicoView source and coupled to a nanoAcquity UPLC. For protein identification and quantification, raw data were processed with FragPipe (V16) having at least two peptides per protein. Protein intensities were reported in the combined_protein.txt file generated by the FragPipe: These were Log_2-transformed and internally normalised against a group of peptides found in all samples, The normalised protein abundance at SOT was subtracted from the abundance at EOT for each patient, A probabilistic dropout model was fitted to the data to estimate fold changes between the treatment and placebo groups at EOT, The proteins were ranked using the t-statistic and a gene-set enrichment analysis was performed to determine the gene sets significantly affected by the treatment compared with the placebo group. Results Gene-ontology analysis revealed that treatment with 16 mg of Nefecon® led to a significant enrichment of multiple pathways (n = 57) involved in several processes previously shown to be important in the pathogenesis of kidney injury in IgAN. These included epigenetic pathways (n = 13), microvesicle formation (n = 3), kidney remodelling (n = 9), and regulation of local immune and inflammatory responses (n = 32). Conclusion We have previously shown that 16 mg of Nefecon® for 9 months positively impacts on multiple biomarkers of IgAN severity. These urine proteomic data support the positive impact of Nefecon® on downstream pro-inflammatory and profibrotic pathways within the kidneys. These data will be validated in the biomarker analyses currently underway as part of the NefIgArd study.