The results of the CRD tests and proteinuria levels in studied groups.

The values of congophilia observed in the paper (A) and membrane (B) tests, as well as the levels of proteinuria (C), are shown. The box plots present the 25th and 75th percentiles (box), the maximum and minimum values, the median (line in the box), and the mean (cross). AN, amyloid nephropathies; Control, patients without proteinuria; CRA, Congo red area; CRR, Congo red retention; NA, non-amyloid nephropathies; NP, normal pregnancy; PE, preeclampsia.</p

The quantitative relationship between urine congophilia and proteinuria in three groups with nephropathies.

The Spearman rank-order correlation coefficients (Spearman’s correlation), 95% confidence intervals (C. I.), p values (t-test), and scatter charts with linear trend lines are shown for each group. AN, amyloid nephropathies; CRR, Congo red retention; NA, non-amyloid nephropathies; PE, preeclampsia.</p

Congophilia of urine samples and HSA solution after centrifugation on concentrators with cut-offs of 30 and 100 kDa.

(A) Samples before centrifugation (original samples) and after centrifugation (concentrates and filtrates) are analyzed using a membrane test. At the left, urine protein fractions are listed. (B) 10% polyacrylamide gel electrophoresis of urine samples with 15 μg of protein is shown. Proteins in the gel are stained by Coomassie brilliant blue. AL, Immunoglobulin light chain amyloidosis; HSA, human serum albumin; MW, molecular weight; PE, preeclampsia.</p

Demographic and clinical data in studied groups.

Preeclampsia is a disorder that can occur during pregnancy and is one of the leading causes of death among pregnant women. This disorder occurs after the 20th week of pregnancy and is characterized by arterial hypertension, proteinuria, fetoplacental, and multiple organ dysfunctions. Despite the long history of studying preeclampsia, its etiology and pathogenesis remain poorly understood, and therapy is symptomatic. One of the factors of the disorder is believed to be misfolded proteins that are prone to form amyloid aggregates. The CRD tests, utilizing the binding of the amyloid-specific dye Congo red to urine components, demonstrate high efficiency in diagnosing preeclampsia. However, these tests have also been found to be positive in other disorders with proteinuria, presumably associated with concomitant amyloidosis. To assess the limitations of the CRD tests, we examined urine congophilia and protein components mediating Congo red positivity in patients with proteinuria, including preeclampsia, amyloid and non-amyloid nephropathies. We stained the urine samples and calculated congophilia levels. We also assessed the contribution of large protein aggregates to congophilia values using ultracentrifugation and determined the molecular weights of congophilic urinary proteins using centrifugal concentrators. All proteinuric groups demonstrate positive results in the CRD tests and congophilia levels were more than two times higher compared with the control non-proteinuric groups (p rs = 0.76), non-amyloid nephropathies (rs = 0.90), and preeclampsia (rs = 0.90). Removal of large aggregates from urine did not affect the congophilia levels. Separation of urine protein fractions revealed congophilic components in the range of 30–100 kDa, including monomeric serum albumin. Our results indicate limitations of CRD tests in preeclampsia diagnostics in women with renal disorders and underscore the need for further research on the mechanisms of Congo red binding with urine components.</div

Congophilia and protein composition in concentrated urine samples from women with uncomplicated pregnancy.

(A) From left to right: initial protein concentrations, protein amounts and CRRs for the four control urine samples (NP/130-134); procedure scheme; protein concentrations and total amounts of protein (weights and percentages of the initial amount) in the supernatant obtained by concentrating. (B) Mean CRRs of concentrated samples and HSA solutions at concentrations equal to concentrated samples are shown. (C) 10% polyacrylamide gel electrophoresis results for original and concentrated samples (1 μg of protein) are shown. BSA (2 μg) was applied to localize the putative HSA in the analyzed samples. Proteins in the gel are stained by Coomassie brilliant blue. BSA, bovine serum albumin; CF, centrifugation; CRR, Congo red retention; NP, control sample from a woman with uncomplicated pregnancy; RT, room temperature.</p

All images used to generate the figures for this study.

All images used to generate the figures for this study.</p

The quantitative relationship between congophilia and concentrations of BGG and HSA in comparison with preeclampsia samples.

The reliability of the linear approximation (coefficient of determination, R2) of CRR dependence on concentrations of BGG (A) and HSA (B) is shown. Section B also shows urinary congophilia in the PE group (grey circles). (C) The ratios of the CRRs to the protein concentrations in HSA solutions and PE samples are shown. CRR, Congo red retention; BGG, bovine gamma globulin; HSA, human serum albumin; PE, preeclampsia.</p

Congophilia of urine samples from patients of different groups before and after ultracentrifugation.

On the left, sample preparation conditions, mean CRRs (n = 11), and 95% C. I. are indicated. AA, serum amyloid A amyloidosis; AL, Immunoglobulin light chain amyloidosis; ANCA, anti-neutrophil cytoplasmic antibody-associated glomerulonephritis; CF, centrifugation; CRR, Congo red retention; IgA, Immunoglobulin A nephropathy; MIgN, monoclonal immunoglobulin-related kidney disease; MN, membranous nephropathy; PE, preeclampsia.</p

The representation of the CRD tests’ results.

The CRD membrane (A) and CRD paper (B) tests are shown. The representative images displayed are from five pregnant women with preeclampsia (1–5) and five control pregnant women (6–10).</p

Experimental data.

All data used to generate the results for this study. (XLSX)</p