Amyloidoses is a group of diseases characterized by the accumulation of abnormal proteins (called amyloids) in different organs and tissues. For systemic amyloidoses, the disease is related to increased levels and/or abnormal synthesis of certain proteins in the organism due to pathological processes, e.g., monoclonal gammopathy and chronic inflammation in rheumatic arthritis. Treatment of amyloidoses is focused on reducing amyloidogenic protein production and inhibition of its aggregation. Therapeutic approaches critically depend on the type of amyloidosis, which underlines the importance of early differential diagnostics. In fact, the most accurate diagnostics of amyloidosis and its type requires analysis of a biopsy specimen from the disease-affected organ. However, absence of specific symptoms of amyloidosis and the invasive nature of biomaterial sampling causes the late diagnostics of these diseases, which leads to a delayed treatment, and significantly reduces its efficacy and patient survival. The establishment of noninvasive diagnostic methods and discovery of specific amyloidosis markers are essential for disease detection and identification of its type at earlier stages, which enables timely and targeted treatment. This review focuses on current approaches to the diagnostics of amyloidoses, primarily with renal involvement, and research perspectives in order to design new specific tests for early diagnosis.

Background and Aims Being a well-known intrinsic property of amyloid, сongophilia was recently demonstrated in urine of patients with proteinuria of different etiology including renal amyloidosis (RA) and non-amyloid nephropathies (NANP) [1]. Urine proteins (UPs) responsible for congophilia in RA and NANP are supposed to have another property of amyloid such as resistance to ionic detergents and present in urine as detergent-resistant aggregates (DRA). In the pilot study we performed mass-spectrometry (MS) analysis of congophilic urine samples and its detergent-resistant fraction in patients with RA and NANP to investigate UPs and their specificity to the particular kidney disease. Method We collected first morning void urine samples from patients with RA (n = 4) and NANP (n = 4). Urine congophilia was assessed by Congo red Dot test as described previously [1]. To analyze bulk urine (BU) proteins, 30 μl of supernatant was precipitated with 80% acetone and boiled in 2% sodium dodecyl sulfate (SDS) for 30 minutes. Then 5 μg of UPs was separated in 10% polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. The ultracentrifugation of 0.5-3 ml of urine at 300,000x g for 16h following by the treatment of precipitate with 3% sarcosyl in phosphate-buffered saline (PBS) for 10 min with subsequent washing of resistant aggregates in PBS were applied to obtain DRA. After boiling in SDS 8 μl of the sample was separated in 10% PAGE. UPs concentration in the DRA was estimated by densitometry regarding to 2 μg of bovine serum albumin. Then 6 μg of UPs was digested with trypsin, followed by purification on silicate (CDS Empore™ C18 Extraction Disks). Prepared samples of BU and DRA were analyzed by electrospray ionization tandem MS. We used MsFragger software to obtain lists of UPs for each sample. Most representative UPs in the sample were selected by the unique spectral count (USC). We considered the protein as having diagnosis-specific potential (DSP) if it was present in every sample of the particular disease group, i.e. AA, AL, NANP in BU or DRA, and was absent in any sample of another disease group. Results The patients had following characteristics: age 51±13 years, 3 male/ 5 female, eGFR = 45 (19; 95) ml/min/1.73 m2; 24 h proteinuria = 6.5 (4.5; 7.9) g. In PAGE analysis, BU proteins appeared to be similar in all 8 samples with 2 major bends in 70 kDa and 50 kDa regions, corresponding to albumin and immunoglobulin (Ig) heavy chains, respectively (Fig. 1A). The amount of protein in DRA was small and comprised 1.02 (0.71; 1.61) % of the total protein in the sample (4.7 (2.3; 5,1) g/l). Compared with BU PAGE analysis of DRA proteins revealed other bends with trace albumin bend, predominance of 45 kDA region bend and more apparent 30 kDa bend (Ig light chains) in the majority of patients (Fig. 1B). Results of MS analysis are shown on the Figure 2. There were more DSP revealed in DRA vs BU: 14 vs 5, 46 vs 2 and 4 vs 2 UPs in samples of AL, AA and NANP, respectively. When compare by a particular disease group, there were no DSP found either in BU or DRA in RA samples. One protein (aminopeptidase N) and 3 proteins (isoform 3 of unconventional myosin-LC, protein S100-A8, elongation factor 1-α 1) were detected as DSP in BU and DRA in AL, respectively. Alpha-1-acid glycoprotein 1 was only DSP for AA in DRA as well as serum paraoxonase 1 for NANP in BU. Venn-diagrams of shared and divergent UPs are present on the Figures 2D-G. Conclusion Although DRA represented a small portion of UPs, its composition significantly differed from BU and could contain more specific disease markers that makes urine detergent-resistant fraction promising for the future research. Understanding the role of DRA proteins in the pathogenesis of amyloid and non-amyloid renal disease and their diagnostic utility requires further studies.

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Background and Aims Amyloidosis is a serious mostly systemic disease caused by the deposition of abnormal proteins with a cross-β-sheet conformation in tissues and predominantly affecting kidneys among other organs that are associated with inferior renal outcome. Nephrotic syndrome is a common clinical manifestation of renal amyloidosis (RA) among other primary glomerulopathies. Nowadays there is a lack of non-invasive diagnostic tools for screening RA. The aim of this pilot study was to investigate the Congo Red Dot (CRD) test supplemented with polarizing microscopy as a non-invasive technique for detecting RA in patients with nephrotic syndrome. Method In this cross-sectional study, we enrolled patients with nephrotic syndrome (NS, n = 41, aged 52±12 years, 48.8% of males) and healthy individuals without proteinuria for control group (CG, n = 32, aged 45±18 years, 45.5% of males). The first-morning urine samples from all included persons were used for the CRD test. In all cases the protein-creatinine ratio (PCR) was assessed at 24-hour urine collection as well as estimated glomerular filtration rate (eGFR) using CKD-EPI formula. Kidney biopsy was carried out for morphological verification of diagnosis in all patients with nephrotic syndrome. For the Congo Red Dot test, 4 µl of Congo red dye solution (CR) was mixed with 2 µl of a sample of the patient's first-morning urine sample. This mixture was applied in duplicates onto a nitrocellulose membrane (0.45 µm). To quantify the Congo Red Retention (CRR, %), which means the staining intensity the membrane was washed in ascending series of ethanol concentrations and photographed under constant conditions before and after washing in the specially designed photo camera. The CRR was determined as the ratio of the average brightness for two spots on the membrane after and before washing in ethanol and calculated using the ImageJ software. For polarizing microscopy urine samples were centrifuged at 1500 g for 15 minutes at +4°C, the supernatant was removed, 5 µl of CR was added to the sediment and 10 µl was applied to a histological slide, and viewed under polarized light. Data are presented as median and interquartile range (M (25%; 75%)). To assess the significance of differences between groups, the nonparametric Mann-Whitney test was used. Spearman's correlation analysis was used to investigate the relationships between the variables. Receiving operating characteristic (ROC) curve was generated to assess the utility of CRR in prediction of RA. Results The CRR in the NS group (42.2% (26.8; 74.2)) significantly differs from the CRR in the CG group (0.0%), p<0.001. PCR in the NS group was 4.8 g/g (2.2; 11.2). The examples of CRD test results are shown in Figure 1a. By morphological diagnosis, patients were divided into two groups: renal amyloidosis (RA, n = 16, aged 58.3±7.5 years, 50% of males) and non-amyloid variant of nephropathy (NN, n = 25, aged 48.6±12.1 years, 48% of males). There was no significant difference between CRR in RA (71.6% (15.0; 91.8)) and NN (41.7% (27.5; 70.4), p=0.41 as well as between PCR (6.5 g/g (1.4; 15.4) in RA and 3.6 g/g (1.9; 10.4) in NN, p=0.34). Correlation between CRR and PCR in the NN (r=0.722, p<0.0001) and RA (r=0.605, p=0.013) was determined. ROC-analysis showed that threshold value for the diagnosis of RA can be taken as CRR=33.5% with sensitivity 68.8% and specificity 68.4% (Figure 1b). Despite the absence of differences in the CRR, polarization microscopy showed specific apple-green birefringence of aggregates bound with CR in the sample of patient with AL-amyloidosis (Figure 2 a, b) contrary to the sample of patient with IgA-nephropathy (Figure 2 c, d). Conclusion Preliminary results suggest that CRD test supplemented with polarizing microscopy could be used for RA screening in patients with nephrotic syndrome but require further validation as far as congophilia of urine samples from patients with non-amyloid nephropathy also presents.