Diagnosing Polyomavirus Nephropathy without a Biopsy: Validation of the Urinary PyV-Haufen-Test in a Proof-of-Concept Study including Uromodulin Knock-out-Mice.

BACKGROUND: Polyomavirus 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 three-dimensional PyV aggregates in the urine, might provide crucial diagnostic information. METHODS: A multistep experimental design. Hypothesis: PyV-haufen form within the kidneys under high concentrations of uromodulin, a kidney specific protein; PyV-haufen are kidney-specific-disease-markers. RESULTS: Investigative step A showed colocalization of uromodulin with aggregated PyV (i) in ten kidneys with PyVN by immunohistochemistry, (ii) in urine samples containing PyV-haufen by electron microscopy/immunogold labeling (n = 3), and (iii) in urine samples containing PyV-haufen by immunoprecipitation assays (n = 4). Investigative step B: In in-vitro experiments only high uromodulin concentrations of ≥ 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). Investigative step C: 0/11 (0%) uromodulin KO-/- mice with histologic signs of PyVN showed urinary PyV-haufen shedding compared to 10/14 (71%) WT+/+ mice. CONCLUSION: PyV-haufen form within kidneys under high uromodulin concentrations. Thus, PyV-haufen detected in the urine are specific biomarkers for intra-renal disease, i.e. definitive PyVN.

The Urinary Polyomavirus-Haufen Test: A Highly Predictive Non-Invasive Biomarker to Distinguish "Presumptive" from "Definitive" Polyomavirus Nephropathy: How to Use It-When to Use It-How Does It Compare to PCR Based Assays?

"Definitive" biopsy proven polyomavirus nephropathy (PyVN), usually caused by BK polyomavirus (BKPyV), remains a significant infection of kidney transplants. Diagnosis depends upon an allograft biopsy and outcome depends upon early intervention. Here, we report data on a non-invasive biomarker for PyVN, the urinary PyV-Haufen test. Test results were compared to those of conventional laboratory assays targeting PyV replication, i.e., BKPy-viremia, -viruria and urinary decoy cell shedding. Of 809 kidney transplant recipients, 228 (28%) showed PyV replication with decoy cell shedding and/or BKPy-viremia by quantitative PCR; only a subset of 81/228 (36%) showed "definitive" PyVN. Sensitivity and specificity for identifying patients with PyVN was: 100% and 98%, respectively, urinary PyV-Haufen test; 50% and 54%, respectively, urinary decoy cell shedding; 97% and 32%, respectively, BKPy-viremia with cut-off of ≥250 viral copies/mL; 66% and 80%, respectively, for BKPy-viremia ≥104 viral copies/mL. The PyV-Haufen test showed a very strong correlation with the severity of PyVN (Spearman's ρ = 0.84) and the Banff PyVN disease classes (p < 0.001). In comparison, BKPy-viremia and -viruria levels by PCR displayed modest correlations with PyVN severity (Spearman's ρ = 0.35 and 0.36, respectively) and were not significantly associated with disease classes. No association was found between decoy cell shedding and PyVN severity or disease classes. Pilot data demonstrated that PyVN resolution with decreasing Banff pvl-scores was reflected by a gradual decrease in PyV-Haufen shedding; such a tight association was not noted for BKPy-viremia. In conclusion, urinary PyV-Haufen testing is a highly specific, non-invasive method to accurately diagnose patients with "definitive" PyVN and to optimize patient management. Assay specifics are discussed.

Glomerular disease frequencies by race, sex and region: results from the International Kidney Biopsy Survey.

Background: Large-scale studies comparing glomerular disease frequencies across continents are lacking. Methods: We surveyed 29 nephropathology laboratories in four continents using a standardized data collection form. We obtained recent consecutive kidney biopsy diagnosis frequencies at each center and summary demographics for each diagnosis. This report focuses on glomerular disease frequencies by region and race/ethnicity. Results: Among 42 603 glomerular disease diagnoses reported (median age 47 years, 52% male, 57% white), from a total of 60 340 diagnoses, glomerular disease subtype frequencies differed considerably by continent. Diabetic glomerulosclerosis (GS; 19.1%) and focal segmental glomerulosclerosis (FSGS; 19.1%) predominated in North America; lupus nephritis (38.1%) and FSGS (15.8%) predominated in Latin America; IgA nephropathy (IgAN; 22.1%) and FSGS (14.9%) predominated in Europe; and IgAN (39.5%) and lupus nephritis (16.8%) predominated in Asia. After stratifying by race, diabetic GS (17.4% versus 4.3%, P < 0.001) and FSGS (17.3% versus 11.8%, P < 0.001) were more, and lupus nephritis less (15.8% versus 45.6%, P < 0.001), frequent among Latinos in North versus Latin America; FSGS was more (13.1% versus 7.1%, P < 0.001), and IgAN less (27.4% versus 40.5%, P < 0.001), frequent among Asians in North America versus Asia; and FSGS (18.9% versus 13.5%, P < 0.001) and diabetic GS (18.7% versus 6.5%, P < 0.001) were more, and IgAN less (14.4% versus 25.4%, P < 0.001), frequent among whites in North America versus Europe. Conclusions: We determined that glomerular disease frequencies differed by continent, even among patients of similar race/ethnicity. Regional environmental and lifestyle factors, and local biopsy policies, might influence glomerular disease epidemiology independently of race/ethnicity.

Cytochrome P450 3A5 expression in the kidneys of patients with calcineurin inhibitor nephrotoxicity.

BACKGROUND: Nephrotoxicity secondary to calcineurin inhibitors is common in renal transplant recipients, occurring in 76-94% of patients. The role of drug transporters (P-glycoprotein) and drug metabolizing enzymes (cytochrome P450) as predisposing factors toward nephrotoxicity or its prevention has not been thoroughly examined. METHODS: The objective of this study was to analyse cytochrome P450 3A5 (CYP3A5) expression in kidneys of solid organ recipients by immunohistochemistry to determine if there is an association between expression of this enzyme and calcineurin inhibitor toxicity. Transplant recipients were compared with a control group. RESULTS: Apical tubular plasma membrane staining for CYP3A5 was present in 62% of study and 100% of control biopsies (P=0.0012). Proximal and distal tubular nuclear staining intensity was similar between groups. Cytoplasmic staining in both the proximal (2.1+/-0.9 vs 1.4+/-0.9) and distal (2.8+/-0.5 vs 1.8+/-1.1) tubules was greater in the control vs study population specimens, respectively (P=0.0093 and P=0.0005, respectively). Regression models that controlled for use of CYP3A inhibiting and inducing medications, age, gender, race and glomerular filtration rate did not predict differences between study groups with regard to staining locations and intensity, except for the cytoplasm of the distal tubule, where intensity of staining was significantly lower in the study group (0.9+/-0.3; P=0.002). CONCLUSIONS: This study showed decreased expression of CYP3A5 in nephrotoxic biopsies as compared with a control group. Our data suggest that the relationship between reduced presence of CYP3A5 in the kidney tubules and nephrotoxicity should be further explored to elucidate the role of this enzyme in mediating toxicity.

Negative-staining electron microscopy of the urine for the detection of polyomavirus infections.

Negative-staining electron microscopy (EM) has played a pivotal role in diagnostic virology. It is a rapid technique for viral detection in the urine and can provide an easy means for monitoring viral activity and productive infections. EM of urine for the detection of polyomaviruses has hitherto not been systematically evaluated as a screening tool for renal transplant patients at risk for BK polyomavirus nephropathy (BKN). Here, the authors discuss technical aspects of negative-staining EM of urine (n = 76 samples) and present a simple and rapid protocol for the semiquantitative evaluation of patient samples. In two patient populations (either with (n = 15 samples) or without (n = 15 samples) an established diagnosis of BKN), EM results were compared with two previously established techniques for monitoring polyomavirus activation: (1) cytology for the quantitation of decoy cells, and (2) quantitative PCR assays for the detection of BK virus DNA load levels. In both patient groups, the dynamics of decoy cell shedding by urine cytology closely paralleled free viral particle shedding by EM, and viral load levels as measured by PCR. A trend toward higher readings was observed in patients with BKN (median values, control versus BKN groups: decoy cells 21 versus 50/slide; free virions by EM: 32 versus 66 viral particles/10 high-power fields; PCR: 3.5 x 10(8) versus 5.4 x 10(8) BK virus copies/ml; all differences not statistically significant). The authors conclude that negative-staining EM and the semiquantitative assessment of free viral particles in the urine can be a useful clinical method to identify patients at increased risk for BKN. EM can be used alone or in combination with urine cytology or PCR assays.

Calcineurin inhibitor-induced nephrotoxicity and renal expression of P-glycoprotein.

STUDY OBJECTIVE: To evaluate immunohistochemistry staining patterns for P-glycoprotein (P-gp) and a marker of early apoptosis (active caspase-3) in renal biopsy specimens obtained from solid organ transplant recipients with nephrotoxicity and those from a control group. DESIGN: Retrospective analysis of pathology specimens and medical records. SETTING: Medical university. SUBJECTS: Twenty-nine solid organ transplant recipients with nephrotoxicity and 32 control patients. MEASUREMENTS AND MAIN RESULTS: Medical records were reviewed for patient demographics, clinical laboratory results, and prescribed drugs. Immunohistochemistry techniques using primary antibodies to P-gp and active caspase-3 were performed to evaluate staining patterns of these proteins in the kidney specimens. Differences in measures of interest between groups were compared with the Fisher exact test for categoric data and Wilcoxon rank sum test for continuous data. Logistic and linear modeling were used to evaluate difference in measures of P-gp and active caspase-3 between groups while controlling for confounders. Immunohistochemistry confirmed the presence of P-gp in the renal tubules (apical and basal membranes and cytoplasm). Intensity of P-gp staining (score range 0-4) was reduced in renal specimens of transplant recipients with nephrotoxicity compared with the control specimens (mean +/- SD intensity scores 3.2 +/- 0.7 vs 3.8 +/- 0.4, p=0.0002). Neither P-gp-inducing nor P-gp-inhibiting drugs predicted expression of P-gp in the renal specimens of either group. The extent of tubular staining (score range 1-4) for the apoptosis marker, active caspase-3, was less in the nephrotoxicity group than in the control group (mean +/- SD extent scores 1.7 +/- 0.6 vs 2.8 +/- 0.5, p=0.0003). CONCLUSION: P-glycoprotein expression was less pronounced in renal biopsy specimens with calcineurin inhibitor-induced nephrotoxicity compared with the nonnephrotoxic control specimens. Reduced P-gp expression was evident even when the analysis controlled for factors such as renal function, age, sex, race, diabetes mellitus, level of proteinuria, or prescribed therapy with P-gp inducers or inhibitors. Interpretation of the results from active caspase-3 staining requires further study.