A pilot study of anti-nephrin antibodies in podocytopaties among adults.

AIM: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are podocytopathies characterized by damage to the glomerular filtration barrier, leading to proteinuria and nephrotic syndrome. The production of anti-podocyte antibodies has been proposed as potential circulating factors contributing to the development of these conditions. The aim of the study is to evaluate the levels of anti-nephrin antibodies in patients with podocytopathies and healthy subjects. METHODS: In this study, a total of 77 patients with active glomerulopathy and 11 healthy subjects were included. Forty one patients were diagnosed with FSGS, 11 with MCD, and 25 with MN. To measure the levels of anti-nephrin antibodies, enzyme-linked immunosorbent assay was used. RESULTS: The levels of antibodies to nephrin were significantly higher in patients with MCD 61.2 [28.9-66.3] ng/mL and FSGS 32.5 [17.2-58.4] ng/mL compared to MN 20.3 [14.4-38.4] and healthy individuals 15.3 [12-18.9] ng/mL, p < .05. In patients with primary FSGS, the levels of antibodies to nephrin were significantly higher 45.2 [20-64.3] ng/mL compared to patients with secondary FSGS 26.7 [11.2-44.1] ng/mL, p < .05. There were no significant differences in the remission rate between the anti-nephrin antibodies positive and negative groups (log-rank test: p = .158). CONCLUSION: The level of anti-nephrin antibodies was found to be significantly higher in patients with MCD and pFSGS compared to those with sFSGS, MN, and healthy subjects. Anti-nephrin antibodies in MCD and primary FSGS may be associated with the severity of podocytopathies, however they did not have an impact on the response to therapy.

Preliminary study of anti-CD40 and ubiquitin proteasome antibodies in primary podocytopaties.

Background: Minimal change disease and focal segmental glomerulosclerosis are primary podocytopathies that are clinically presented in adults presenting with severe nephrotic syndrome. The pathogenesis of these diseases is not clear and many questions remain to be answered. A new concept about the role of changes in the antigenic determinant of podocytes and the production of anti-podocyte antibodies that cause podocyte damage is being developed. The aim of the study is to evaluate the levels of anti-CD40 and anti-ubiquitin carboxyl-terminal hydrolase L1 (anti-UCH-L1) antibodies in patients with podocytopathies in comparison with other glomerulopathies. Methods: One hundred and six patients with glomerulopathy and 11 healthy subjects took part in the study. A histological study revealed primary FSGS in 35 patients (genetic cases of FSGS and secondary FSGS in the absence of NS were excluded), 15 had MCD, 21 - MN, 13 - MPGN, 22 patients - IgA nephropathy. The effect of steroid therapy was evaluated in patients with podocytopathies (FSGS and MCD). The serum levels of anti-UCH-L1 and anti-CD40 antibodies were measured by ELISA before steroid treatment. Results: The levels of anti-UCH-L1 antibodies were significantly higher in MCD patients and anti-CD40 antibodies were higher in MCD and FSGS than in the control group and other groups of glomerulopathies. In addition, the level of anti-UCH-L1 antibodies was higher in patients with steroid-sensitive FSGS and MCD, and anti-CD40 antibodies were lower than in patients with steroid-resistant FSGS. An increase in anti-UCH-L1 antibody levels above 6.44 ng/mL may be a prognostic factor of steroid-sensitivity. The ROC curve (AUC = 0.875 [95% CI 0.718-0.999]) for response to therapy showed a sensitivity of 75% and specificity of 87.5%. Conclusion: An increase in the level of anti-UCH-L1 antibodies is specific for steroid-sensitive FSGS and MCD, while an increase in anti-CD40 antibodies - for steroid-resistant FSGS, compared with other glomerulopathies. It suggests that these antibodies could be a potential factor for differential diagnosis and treatment prognosis.

CD44 Expression in Renal Tissue Is Associated with an Increase in Urinary Levels of Complement Components in Chronic Glomerulopathies.

It is suggested that activated CD44+ cells play a profibrogenic role in the pathogenesis of active glomerulopathies. Complement activation is also involved in renal fibrogenesis. The aim of the study was to evaluate the role of the activation of CD44+ cells in the kidney tissue and complement components' filtration to the urine as factors of renal tissue fibrosis in patients with glomerulopathies. In total, 60 patients with active glomerulopathies were included in our study: 29 patients with focal segmental glomerulosclerosis (FSGS), 10 patients with minimal change disease (MCD), 10 patients with membranous nephropathy (MN), and 11 patients with IgA nephropathy. The immunohistochemical peroxidase method was used to study the expression of CD44+ in kidney biopsies. Components of complement were analyzed in urine by the multiple reaction monitoring (MRM) approach using liquid chromatography. Strong CD44 expression was noted predominantly in PEC and mesangial cells (MC) in patients with FSGS, and to a lesser extent, in patients with MN and IgA nephropathy, and it was absent in patients with MCD. Expression of profibrogenic CD44+ in glomeruli correlated with the levels of proteinuria and complement C2, C3, and C9 components, and CFB and CFI in urine. The CD44+ expression scores in the renal interstitium correlated with the level of C3 and C9 components of complement in the urine and the area of tubulo-interstitial fibrosis. The strongest expression of CD44+ was found in the glomeruli (MC, PEC, and podocytes) of patients with FSGS compared with other glomerulopathies. The CD44 expression score in the glomeruli and interstitium is associated with high levels of complement components in the urine and renal fibrosis.

Targeted MRM Quantification of Urinary Proteins in Chronic Kidney Disease Caused by Glomerulopathies.

Glomerulopathies with nephrotic syndrome that are resistant to therapy often progress to end-stage chronic kidney disease (CKD) and require timely and accurate diagnosis. Targeted quantitative urine proteome analysis by mass spectrometry (MS) with multiple-reaction monitoring (MRM) is a promising tool for early CKD diagnostics that could replace the invasive biopsy procedure. However, there are few studies regarding the development of highly multiplexed MRM assays for urine proteome analysis, and the two MRM assays for urine proteomics described so far demonstrate very low consistency. Thus, the further development of targeted urine proteome assays for CKD is actual task. Herein, a BAK270 MRM assay previously validated for blood plasma protein analysis was adapted for urine-targeted proteomics. Because proteinuria associated with renal impairment is usually associated with an increased diversity of plasma proteins being present in urine, the use of this panel was appropriate. Another advantage of the BAK270 MRM assay is that it includes 35 potential CKD markers described previously. Targeted LC-MRM MS analysis was performed for 69 urine samples from 46 CKD patients and 23 healthy controls, revealing 138 proteins that were found in ≥2/3 of the samples from at least one of the groups. The results obtained confirm 31 previously proposed CKD markers. Combination of MRM analysis with machine learning for data processing was performed. As a result, a highly accurate classifier was developed (AUC = 0.99) that enables distinguishing between mild and severe glomerulopathies based on the assessment of only three urine proteins (GPX3, PLMN, and A1AT or SHBG).

A cross-sectional study of antibodies to ubiquitin proteasome system in different glomerulopathies.

BACKGROUND: Recently, evidence has emerged that the ubiquitin system, which is involved in extracellular protein degradation, is most susceptible to damage in podocytes in cases of podocytopathies. We studied anti-ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) antibodies in glomerulopathies with proteinuria. MATERIALS AND METHODS: 71 patients with glomerulopathy and 11 healthy subjects were included in our study. 44 patients had nephrotic syndrome, and 27 did not. Serum levels of anti-UCHL1 antibodies were measured by ELISA. RESULTS: The levels of anti-UCHL1 antibodies were significantly higher in focal segmental glomerulosclerosis (FSGS) patients than in minimal change disease (MCD), IgA nephropathy, membranous nephropathy, or membranoproliferative glomerulonephritis patients and control group. The levels of UCHL1 antibodies in serum did not correlate with 24-hour proteinuria, blood pressure, glomerulosclerosis percentage, or area of tubulointerstitial fibrosis, but did correlate with serum creatinine and estimated glomerular filtration rate (eGFR). During the development of the ROC curve (AUC = 0.766 (95% CI 0.634 - 0.897)) for FSGS vs. other forms of glomerulopathies, a readjustment of the sensitivity of 75% and specificity of 61% were established. A former increase in anti-UCHL1 antibody levels above 1.93 ng/mL may be a marker of FSGS OR 3.617 (95% CI 1.051 - 12.447), p = 0.041. CONCLUSION: An increase in the level of anti-UCHL1 antibodies in the serum was noted in FSGS, which suggests that these antibodies could be a potential biomarker for FSGS patients.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Serum levels of plasminogen activator urokinase receptor and cardiotrophin-like cytokine factor 1 in patients with nephrotic syndrome.

The pathogenesis of primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) remains unknown to date. Some circulating permeability factors are being discussed. This work assessed molecule candidates for permeability in serum samples of patients with nephrotic syndrome (NS). MATERIALS AND METHODS: 41 patients with chronic glomerulonephritis (CGN) were included in our study. 17 patients had FSGS, 7 patients had MCD, 5 patients had membranoproliferative glomerulonephritis (MPGN), 6 patients had IgA nephropathy, and 6 patients had membranous nephropathy (MN). The laboratory data were compared with the clinical and histological features of nephritis. Serum levels of plasminogen activator urokinase receptor (uPAR) and cardiotrophin-like cytokine factor 1 (CLCF-1)were measured by ELISA. RESULTS: The serum levels of uPAR were higher in FSGS patients before treatment than in patients with other morphological forms (MCD, IgA nephropathy, MN, and MPGN). The levels of uPAR in serum did not correlate with daily proteinuria, serum creatinine/eGFR, arterial hypertension, the number of sclerosed glomeruli, or tubulointerstitial fibrosis. No correlations were found between the levels of CLCF-1 in serum and creatinine levels/glomerular filtration rate, the percentage of sclerosed glomeruli, or the severity of tubulointerstitial fibrosis. There were no significant differences between the histological variants of nephritis. However, we found correlations between CLCF-1 levels and proteinuria and lipid levels. CONCLUSION: The data indicate an increase in the serum uPAR levels of FSGS before treatment. CLCF-1 levels in serum do not depend on histological forms of CGN, kidney function, or immunosuppressive treatment, but they correlate with proteinuria and serum lipids in patients with NS.

Urinary Protein and Peptide Markers in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a non-specific type of kidney disease that causes a gradual decline in kidney function (from months to years). CKD is a significant risk factor for death, cardiovascular disease, and end-stage renal disease. CKDs of different origins may have the same clinical and laboratory manifestations but different progression rates, which requires early diagnosis to determine. This review focuses on protein/peptide biomarkers of the leading causes of CKD: diabetic nephropathy, IgA nephropathy, lupus nephritis, focal segmental glomerulosclerosis, and membranous nephropathy. Mass spectrometry (MS) approaches provided the most information about urinary peptide and protein contents in different nephropathies. New analytical approaches allow urinary proteomic-peptide profiles to be used as early non-invasive diagnostic tools for specific morphological forms of kidney disease and may become a safe alternative to renal biopsy. MS studies of the key pathogenetic mechanisms of renal disease progression may also contribute to developing new approaches for targeted therapy.

Heat shock protein 90 and NFkB levels in serum and urine in patients with chronic glomerulonephritis.

Heat shock proteins play an important role in immune inflammation and the formation and restoration of proteins. In recent years, the importance of heat shock protein 90 (Hsp90) in the activation of immune inflammation through nuclear factor kB (NFkB) has been discussed. To assess the activation of the Hsp90-NFkB system by measuring serum and urinary levels in patients with chronic glomerulonephritis (CGN). This study included 32 patients with active forms of CGN and 14 patients with Fabry nephropathy. The control group included 10 healthy individuals. Twenty-one out of 32 CGN patients had nephrotic syndrome (NS). Eleven out of 32 CGN patients had proteinuria levels from 1 to 3 g/day without nephrotic syndrome. A total of 17 patients had renal dysfunction (estimated glomerular filtration rate < 60 ml/min/1.73m2). Fourteen patients with Fabry nephropathy had proteinuria without nephrotic syndrome. Serum and urine HSP-90 and NFkB p65 levels were determined using an enzyme-linked immunosorbent assay. The levels of HSP-90 and NFkB in the serum of patients with CGN were significantly higher than in healthy individuals and patients with Fabry nephropathy. In patients with Fabry nephropathy, the HSP-90 and NFkB levels in the urine and serum did not significantly differ from those in the control subjects. Serum Hsp90 levels were significantly higher in the CGN patients with NS than in patients without NS, as well as in patients with normal renal function compared with patients with an eGFR < 60 ml/min/1.73 m2 and patients with tubulo-interstitial fibrosis. Higher levels of HSP-90 and NFkB in serum were observed in patients with nephrotic forms of CGN, including focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy. There were no correlations between the clinical signs of CGN and urinary HSP90/NFkB levels. Activation of the HSP-90-NFkB system, which is directly involved in the development of immune inflammation in CGN, was found in patients with an active course of CGN, especially in those with nephrotic syndrome.