Abstract: FR-PO968

Conversion of Single Glomerular and Tubule Segments into Proteomic Data for Discovery of Pathomechanisms in Proteinuric Kidney Diseases

Session Information

Category: Bioengineering and Informatics

  • 101 Bioengineering and Informatics

Authors

  • Rinschen, Markus M., University Hospital Cologne, Cologne, Germany
  • Huber, Tobias B., University Medical Center Hamburg, Hamburg, Germany
  • Benzing, Thomas, University Hospital Cologne, Cologne, Germany
  • Höhne, Martin, University Hospital Cologne, Cologne, Germany
Background

Diseases of the kidney often originate in the glomerulus. Proteinuria, the leakage of the renal filter, is a hallmark and accelerator of renal disease. Virtually all glomerular diseases are histologically heterogeneous at early stages and affect a certain percentage of glomeruli to different extents. Our understanding of physiological kidney function is largely derived from the functional analysis of single nephron segments.

Methods

Here, we developed a nano-scale sample preparation protocol combined with targeted proteomics and single-unit bioinformatics analysis for the discovery of pathogenetic mechanisms in both single glomeruli and single tubuli. The method was applied to clinical samples, as well as different rodent models of glomerular disease.

Results

We analyzed the proteome of single glomeruli from mice and humans in different podocyte injury models. We could identify up to 2000 proteins (~10000 peptides) per single glomerulus. Analysis of 7 single glomeruli from patients with congenital nephrotic syndrome and proven NPHS1 mutations discovered decreased amounts of nephrin but unchanged podocin amounts. A targeted podocyte sentinel assay was designed to acquire reproducible proteomic data across 24 glomeruli from two different podocyte damage models. The assay monitors podocyte actin cytoskeletal function, slit diaphragm function, glomerular basement membrane abundance and podocyte stress response. Bioinformatic analysis using hierarchical clustering and correlational approaches delineated common responses in glomerular damage across glomerular populations and across models. The method was also able to clearly discriminate microdissected tubular segments (e.g. S1 proximal tubule, mTAL and cortical collecting ducts), with potential applications in renal physiology.

Conclusion

These data demonstrate the potential of single segment proteomics for both basic research and translational medicine.

Funding

  • Government Support - Non-U.S.