Proteomic studies in endemic nephropathy.

Endemic nephropathy (EN) is a chronic tubulointerstitial nephropathy with an early insidious and slow development into terminal renal failure. Proteomics is the systematic study of a proteome, which is the total protein content of a cell, organism or body fluids. Application of proteomic technologies in nephrology has enabled more detailed analyses of protein functions and examined their importance in various physiological and pathological states. Biomarkers with high specificity and sensitivity to early diagnosis are needed for a better understanding of the mechanisms of EN development and its consequences. Urine beta2-microglobulin (B2M) was mainly used as a tubular marker of EN but recently alpha1-microglobulin (AMBP) was proposed for the diagnosis of EN. We studied the urine proteins of 360 patients with EN, diabetic nephropathy (DN) and acute kidney injury (AKI) and the healthy population using proteomic tools. Protein maps from the urine of patients with EN showed significant differences in comparison to the healthy subjects and patients with DN and AKI. Our study highlights six proteins in urine that were differentially excreted in the urine of EN patients compared with the other groups and have potential to be markers for EN prediction. In one of our studies, using routine biomarkers, we investigated the potential of urine B2M, AMBP, albumin and total protein as diagnostic markers for EN, in comparison to glomerulonephritis, nephrosclerosis and a healthy state. Modern proteomic technologies are still robust investigation tools, but can access a vast amount of information from one set of experiments in comparison to a classic diagnostic approach.

Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies.

[Antiviral/immunomodulatory combination therapy: pegylated interferon alpha 2a and ribavirin in patients with chronic hepatitis C virus infection].

INTRODUCTION: Chronic hepatitis C virus (HCV) infection can progress to liver cirrhosis that causes bleeding from the gastrointestinal tract, liver failure and primary hepatocellular carcinoma. Use of standard therapeutic option consists of recombinant pegylated interferon alpha 2a/b with ribavirin in order to eradicate virus and prevent complications. OBJECTIVE: The aim of investigation was to evaluate efficiency of combination therapy (pegylated interferon alpha 2a/b plus ribavirin) in patients with chronic HCV infection and to estimate predictive factors for successful treatment. METHODS: A total of 387 patients with confirmed diagnosis of hepatitis C were evaluated (aged 18-65 years of both genders). Patients were treated with pegylated interferon alpha 2a/b and ribavirin according to a standard regimen lasting 24 or 48 weeks, dependent on virus genotype. RESULTS: Negative HCV RNA (PCR assay) was recorded in 79.7% of patients at the end of treatment. Six months after completed therapy, negative HCV RNA, i.e. stained virologic response (SVR) was assessed in 70.5% of patients. Statistical summary of our results concerning SVR confirmed better efficiency of combination therapy for the following parameters compared to other investigated variables: age < or = 40 (84.3% vs. 59.1%; p < 0.0005), absence of cirrhosis (75.6% vs. 58.3%; p = 0.003), lack of genotype 1 (86.6% vs. 61.8%; p < 0.0005), and in patients who received full doses of pegylated interferon alpha 2a (78.3% vs. 63.3%; p = 0.002). CONCLUSION: Combination therapy of recombinant pegylated interferon alpha 2a with ribavirin leads to SVR in the majority of treated patients (70.5%). Successful treatment depends on a variety of host and virus factors.

Expression of regulatory proteins and proliferative activity in relation to phenotypic characteristics of upper urothelial carcinoma.

BACKGROUND/AIM: Deregulation of the normal cell cycle is common in upper urothelial carcinoma (UUC). The aim of this study was to investigate the expression of regulatory proteins of the cell cycle (p53, p16, cyclin D1, HER-2) and proliferative Ki-67 activity in UUC, and to determine their interaction and influence on the phenotypic characteristics of UUC. METHODS: In 44 patients with UUC, histopathological and immunohistochemical analyses (p53, p16, cyclin D1, HER-2, and Ki-67) of tumors were done. RESULTS: Overexpression/altered expression of p53, p16, cyclin D1 or HER-2 was detected in 20%, 57%, 64%, and 57% of tumors, respectively. Eleven (25%) UUC had a high proliferative Ki-67 index. Forty patients (91%) had at least one marker altered, while four (9%) tumors had a wild-type status. Analysis of relationship between expressions of molecular markers showed that only high expression of p53 was significantly associated with altered p16 activity (p < 0.05). High Ki-67 index was associated with the high stage (p < 0.005), solid growth (p < 0.01), high grade (p < 0.05), and multifocality p < 0.05) of UUC, while high expression of p53 was associated with the solid growth (p < 0.05). In regression models that included all molecular markers and phenotypic characteristics, only Ki-67 correlated with the growth (p < 0.0001), stage (p < 0.01), grade (p < 0.05) and multifocality (p < 0.05) of UCC; (Ki-67 and HER-2 expression correlated with the lymphovascular invasion (p < 0.05). CONCLUSIONS: This investigation showed that only negative regulatory proteins of the cell cycle, p53 and p16, were significantly associated in UUC, while proliferative marker Ki-67 was in relation to the key phenotypic characteristics of UUC in the best way.

Identification and validation of six proteins as marker for endemic nephropathy.

Endemic nephropathy (EN) is defined as a slow progressive renal tubulointestitial disease that mainly occurs in the restricted areas of the Balkan Peninsula. The complexity of the pathogenesis of EN makes its earlier diagnosis very difficult. Urine samples from healthy volunteers from EN regions, EN patients with proteinuria less than 150 mg/L and EN patients with proteinuria more than 150 mg/L, patients with acute kidney injury, patients with diabetic nephropathy and healthy volunteers from Germany were collected. The urinary proteome analyses were performed using 2-D DIGE and mass spectrometry. The validation of biomarkers was investigated by two approaches (Western blot (WB) and dot blot) in successively increasing size - and partially overlapping - sample sets. Comparative and statistical analyses of the proteomics data from the different patient groups allowed the identification of six proteins (alpha-1-microglobulin, alpha-2-glycoprotein-1, beta-2-microglobulin, mannose-binding-lectin-2, protection-of-telomeres-protein-1, and superoxide-dismutase [Cu-Zn]), which were able to discriminate EN with low and high proteinuria from the other groups with high significance (p<0.05). The reliability of the identified proteins as EN marker was underlined with high statistical significance using WB analyses (sensitivity 66.7-98% and specificity 70-100%), whereas the dot blot analyses revealed a decrease in the sensitivity and specificity of these biomarkers.

Beta2-microglobulin and alpha1-microglobulin as markers of Balkan endemic nephropathy, a worldwide disease.

BACKGROUND: Urine beta2-microglobulin (beta2-MG) was mainly used as a tubular marker of Balkan endemic nephropathy (BEN) but recently alpha1-microglobulin (alpha1-MG) was proposed for the diagnosis of BEN. In this study, the potential of urine beta2-MG, alpha1-MG, albumin, and total protein in the differentiation of BEN from healthy persons and patients with glomerulonephritis (GN) and nephrosclerosis (NS) was examined. METHODS: This study involved 47 patients with BEN, 36 with GN, 11 with NS, 30 healthy subjects from BEN families, and 46 healthy subjects from non-BEN families. RESULTS: In BEN patients area under the curve (AUC) for urine beta2-MG (0.828) and alpha1-MG (0.782) was higher than for urine albumin (0.740), but in GN patients AUC for urine protein (0.854) and albumin (0.872) was significantly higher than for the two low molecular weight proteins. AUC for all four urinary markers in NS patients was significantly lower than in BEN patients, ranging between 500 and 595. Median urine beta2-MG excretion in BEN patients was 17.5 times higher than in GN patients and 18.3 times higher than in controls; median alpha1-MG excretion was higher only 3.0 and 2.25 times, respectively. In the differentiation of BEN from healthy controls, beta2-MG had higher sensitivity and specificity at the cutoff levels (p < 0.001) than alpha1-MG (p < 0.05). In the differentiation of BEN from GN, beta2-MG was the best marker. CONCLUSION: All four urinary markers can be used for the differential diagnosis of BEN, beta2-MG being the best. Like in aristolochic acid nephropathy, beta2-MG seems to be an early marker of tubular damage in BEN.

Short-term increase of glucose concentration in PDS results in extensive removal and high glycation level of vital proteins during continuous ambulatory peritoneal dialysis.

BACKGROUND: The effect of clearance of so-called middle- and high-molecular weight proteins on clinical outcome of patients treated by peritoneal dialysis is still a matter of debate. In our present study, we investigated the impact of short-time alteration of the glucose concentration and the osmolarity of the peritoneal dialysis solution (PDS) on protein removal. METHODS: Peritoneal dialysis liquids (PDL) were collected from 19 well-characterized continuous ambulatory peritoneal dialysis (CAPD) patients treated with two types of PDS: Baxter (n = 10) and Fresenius (n = 9). The patients were treated with two different glucose concentration of each PDS in 4-h cycles. The depletion of the six interfering high-abundant proteins from the PDL samples was performed with the Multiple Affinity Removal LC Column-Human 6. The resulting protein fractions were analysed by 2D gel electrophoresis, differential in gel electrophoresis, mass spectrometry and 2D western blot. RESULTS: Proteomics investigation of the PDL fractions after depletion allowed the identification of 198 polypeptides of 424 excised spots. These polypeptides equates to 48 non-redundant proteins. Comparative analyses of 2D gel electrophoresis protein pattern revealed a clear correlation between protein removal and PDS glucose concentration and osmolarity. An increase for 4 h in the PDS osmolarity (with 43-51 mosmol/L) resulted qualitatively in 18-23% more protein removal in PDL. Moreover, 2D western blot analyses of the protein glycation pattern showed that the short-time increase in PDS glucose concentration (45-50 mM) resulted in significant alteration of the advanced glycosylation end products (AGEs) pattern. CONCLUSIONS: The data presented in this study demonstrate a clear correlation between the short-time changes in glucose concentration and osmolarity of the PDS, and the augmentation of the protein removal and the appearance of AGEs during CAPD.

Divergent squamous differentiation in upper urothelial carcinoma-comparative clinicopathological and molecular study.

Upper urothelial carcinoma (UUC) has a plasticity to demonstrate divergent differentiation with squamous metaplastic elements. There was no previous study exploring profiling of molecular markers in metaplastic squamous upper urothelial carcinoma (SUUC) and conventional upper urothelial carcinoma (CUUC). The aims of this study was to compare expression of the phenotypic characteristics of tumors and molecular markers (p53, p16, cyclin D1, E-cadherin, HER-2, Ki-67, Bcl-2, Bax) in SUUC and CUUC. SUUC was detected in 20% of 44 patients. There was significant difference between SUUC and CUUC in the pathological stage, grade, growth and presence of lymphovasular invasion (p < 0.05; 0.05; 0.05; 0.01 respectively). The mean Ki-67 and p53 labeling index was significantly higher in SUUC than in CUUC (p < 0.05; 0.05). There was no significant difference in the expression of p16, cyclin D1, E-cadherin, HER-2, Bcl-2 and Bax between SUUC and CUUC. Univariant model showed that SUUC was significantly associated with lymphovascular invasion (p = 0.007), Ki-67 activity (p = 0.016) and growth (p = 0.026). Exploration of UUC with squamous divergent differentiation showed changes in phenotypic characteristics and Ki-67, as well as similar molecular profile with CUUC.

Role of stem cells in kidney repair.

End-stage renal disease and acute renal failure are the most important issues of practical and clinical nephrology, bearing in mind their high mortality rate, solely symptomatic treatment, and overall economic impact on society. The advances in stem cell biology opened the door for the new era in treatment of many disorders, including renal, offering new therapeutical solutions. Findings suggesting that the adult kidney contains stem cells and that stem cells from bone marrow have potential to differentiate into renal cells focused research on the possible application of these cells in therapy of kidney disorders. The other promising candidates for stem cell therapy for the kidney are embryonic stem cells and amniotic fluid-derived stem cells. This article focuses on the characteristics and possible application of these types of stem cells.