Urinary Polyomavirus-Haufen Shedding in Mouse and Man: A Proof-of-Concept Study for a Non-Invasive Urine Biomarker for Polyomavirus Nephropathy
Volker Nickeleit, Bruna Brylawski, Harsharan Singh, Lauraine Rivier. University of North Carolina at Chapel Hill, Chapel Hill, NC
Background: Polyomavirus Nephropathy (PVN) is the most significant viral renal allograft infection with 4% incidence. A definitive diagnosis is only established by renal biopsy. Recently we described a novel, non-invasive urinary assay to diagnose PVN, i.e. the "polyomavirus (PV)-Haufen-Test," with positive and negative predictive values of >95% for definitive biopsy confirmed PVN. This test has great diagnostic potential.Hypothesis: 1) PV-aggregation and Haufen formation depend on a high concentration of Tamm-Horsfall protein (THP) that is only present in primary urine found in injured renal tubules. Such THP concentrations are not detected in secreted secondary urine. 2) Urinary PV-Haufen are found in a mouse model of PVN but not in control animals. Thus the presence of PV-Haufen in voided urines is specific for intrarenal disease, and PV-Haufen are reliable diagnostic biomarkers.
Design: Part A - association of PV-Hauden and THP: A1) Immunohistochemistry of PVN cases (n=3) with double labeling to detect PV and THP in renal tubules. A2) Immunogold electron microscopy of urinary PV-Haufen to detect Haufen-bound THP (n=3). A3) Immunoprecipitation studies on urinary PV-haufen (n=6). A4) Dilution curves altering THP concentrations in fluids mimicking primary and secondary urines and evaluating for PV-aggregation and Hafuen formation (n=5).
Part B - THP concentrations in voided urine samples (with concurrent kidney biopsies; n=12).
Part C - Mouse model of PVN: Search for urinary PV-Haufen in mice with PVN (n=5) and control animals without PVN (n=5).
Results: Part A: A1) In PVN, intratubular PV aggregates show abundant THP. A2) Urinary PV-Haufen are intimately admixed with THP. A3) Immunoprecipitation of urinary PV-Haufen shows abundant coprecipitation of PV and THP. A4) PV Aggregation and PV-Haufen formation is THP dose-dependent and only occurs with high THP concentrations of >1mg THP/ml fluid mimicking primary urine in injured tubules. Part B: THP concentrations in voided urine samples are low, median THP 4.5 microgram/ml urine, range: 0.7-19.5.
Part C: All mice with PVN (florid disease stage B) shed abundant PV-Haufen in voided urine samples; PV-Haufen are not detected in control animals.
Conclusions: PV-Haufen formation occurs in the setting of very high THP concentrations found in injured renal tubules exceeding voided urine THP concentrations by 50 fold. A new mouse model of PVN shows typical urinary PV-Haufen in diseased animals. Urinary PV-Haufen are novel dignostic biomarkers for intrarenal PVN.
Category: Kidney (does not include tumors)
Monday, March 4, 2013 1:00 PM
Poster Session II # 225, Monday Afternoon