Diagnosing Polyomavirus Nephropathy Without a Biopsy: Validation of the Urinary Polyomavirus-Haufen Test in a Proof-of-Concept Study Including Uromodulin Knockout Mice

Abstract

Abstract Background Polyomavirus (PyV) nephropathy (PyVN) leads to kidney transplant dysfunction and loss. Since a definitive diagnosis requires an invasive kidney biopsy, a timely diagnosis is often hampered. In this clinical dilemma the PyV haufen-test, centering around the detection of 3-dimensional PyV aggregates in the urine, might provide crucial diagnostic information. Methods A multistep experimental design was used. The hypothesis was that PyV-haufen form within the kidneys under high concentrations of uromodulin, a kidney-specific protein and that PyV-haufen are, therefore, kidney-specific disease biomarkers. Results The first investigative step showed colocalization of uromodulin with aggregated PyV (1) in 10 kidneys with PyVN by immunohistochemistry, (2) in urine samples containing PyV-haufen by electron microscopy/immunogold labeling (n = 3), and (3) in urine samples containing PyV-haufen by immunoprecipitation assays (n = 4). In the in vitro experiments of the next step, only high uromodulin concentrations (≥1.25 mg/mL) aggregated PyV, as is expected to occur within injured nephrons. In contrast, in voided urine samples (n = 59) uromodulin concentrations were below aggregation concentrations (1.2−19.6 µg/mL). In the third investigative step, none of 11 uromodulin−/− knockout mice (0%) with histologic signs of PyVN showed urinary PyV-haufen shedding, compared with 10 of 14 uromodulin+/+ wild-type mice (71%). Conclusions PyV-haufen form within kidneys under high uromodulin concentrations. Thus, PyV-haufen detected in the urine are specific biomarkers for intrarenal disease (ie, definitive PyVN).