Abstract: FR-PO735

A Novel Method of Urine Protein Isolation Allows for Identification of Kidney Disease Protein Biomarkers Using Mass Spectrometry Analysis

Session Information

Category: Glomerular

  • 1004 Clinical/Diagnostic Renal Pathology and Lab Medicine

Authors

  • Shapiro, John P., Ohio State University Wexner Medical Center, Columbus, Ohio, United States
  • Mejia-Vilet, Juan M., Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico, Mexico
  • Birmingham, Daniel J., Ohio State University Wexner Medical Center, Columbus, Ohio, United States
  • Rovin, Brad H., Ohio State University Wexner Medical Center, Columbus, Ohio, United States
Background

Characterization of the urine proteome using mass spectrometry (MS) provides an opportunity to identify kidney disease biomarkers. This approach has proven challenging due to salt content, pH and other physicochemical qualities that interfere with MS. We describe a novel, simple method that attenuates these properties of urine and allows for robust quantitative urine proteome characterization.

Methods

Urine (1ml) from healthy individuals (control) or patients with class IV lupus nephritis (LN-IV) was placed in Nunc MaxiSorp ELISA plates and incubated 16 hr at 4oC. Wells were washed 2x with PBS, 2x with 50 mM NH3HCO3 and incubated with trypsin in 50 mM NH3HCO3 for 7 hours at 37oC. Acetonitrile (ACN) was added to each well, mixed, collected and pooled. Peptides were dried, resuspended in 10 µl 2% ACN, 0.1% formic acid and analyzed by LC-MS/MS on a Thermo-Fisher Orbitrap XL mass spectrometer.

Results

MS identified 356 proteins, 63 of which demonstrated a ≥3-fold change in expression between control and LN-IV (all p<0.05). Several serum proteins such as albumin and serotransferrin were detected at higher levels in LN-IV (27-, 65-fold respectively) than control. In contrast, many proteins known to be expressed by the kidney were present in control urine but were undetectable or significantly decreased in LN-IV urine. Examples include uromodulin, extracellular sulfatase, EGF, kininogen-1, and cubulin, down-regulated (7-, 9-, 12-, 7- and 9-fold, respectively). Other attenuated proteins include amiloride-sensitive sodium channel subunit gamma and sodium/potassium-transporting ATPase subunit gamma, down-regulated 4- and 3-fold, respectively.

Conclusion

This method demonstrates the effects of lupus nephritis on urine protein profiles. As expected, serum proteins prominent in the urine of LN-IV are absent or present in small amounts in control urine. The absence or diminished expression of proteins in LN-IV urine that are present in control urine suggests specific attenuation due to LN-induced renal injury and may provide a way to assess the health of renal parenchyma. This method may provide a rapid means of preparing urine for clinical MS analysis.

Funding

  • Clinical Revenue Support