Insights into ADPKD Pathogenesis from Cyst Fluid Proteomic Profiling

Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the progressive formation and enlargement of fluid-filled cysts, ultimately leading to end-stage renal disease. Despite advances in our understanding of the genetic basis of ADPKD, the molecular composition of cyst fluid- an important medium that may harbor pathogenic signals or biomarkers of cystogenesis remains largely unexplored. Methods: We performed proteomic analysis of pooled cyst fluid samples collected from 10 ADPKD patients (4 males, 6 females). Samples were subjected to tryptic digestion and quenched with formic acid. Depending on protein concentration, 5–10 µL of each sample was loaded onto a C18 reverse-phase column and analyzed using a FAIMS-enabled Orbitrap Ascend mass spectrometer via LC-MS/MS. Peptide spectral data were processed using the SeQuest algorithm in Proteome Discoverer and searched against the human UniProt database. Results: Initial analysis revealed presence of 294 proteins of which albumin constituted approximately 70–75% of the total protein content and immunoglobulins abundantly expressed, indicating plasma proteins. Sex-specific comparisons (male: female) revealed differentially expressed proteins (p-value < 0.05; log2 abundance ratio ≥ 1 or ≤ 1) which were part of apoptosis, humoral immune response, extracellular matrix, G protein couple receptor and inflammatory pathways. To improve the detection of low-abundance proteins, albumin and IgG depletion was performed. This reduced albumin content from ~70–75% to ~20–30%, and decreased IgG levels substantially. Subsequent mass spectrometry analysis of the depleted samples identified 114 unique proteins absent in the non-depleted fraction, while 196 proteins were common to both, and 98 were exclusive to the non-depleted samples. Sex-specific comparisons (male: female) revealed differentially expressed proteins (p-value < 0.05; log2 abundance ratio ≥ 1 or ≤ 1) which were part of macrophage activation, systemic inflammation, hematopoiesis, bone and retinol metabolism pathways. Conclusion: This study provides the first detailed proteomic profile of ADPKD cyst fluid and highlights both novel and previously implicated in ADPKD that may represent new biomarkers or therapeutic targets for cyst growth and progression. Funding: NIDDK Support, Other U.S. Government Support, Private Foundation Support