Quantitative analysis of protein crotonylation identifies its association with immunoglobulin A nephropathy.

Posttranslational modifications (PTMs) to histones such as lysine crotonylation are classified as epigenetic changes. Lysine crotonylation participates in various cellular processes and occurs in active promoters, directly accelerating transcription. The present study performed a proteomics analysis of crotonylation between healthy controls and patients with immunoglobulin A (IgA) nephropathy using tandem mass spectrometry and high­resolution liquid chromatography. The present results identified 353 crotonylated proteins and 770 modification sites, including 155 upregulated and 198 downregulated crotonylated proteins. In total, seven conserved motifs were identified in the present study. The present bioinformatics analysis results suggested a number of the crotonylated proteins exhibited various subcellular localization patterns, such as in the cytoplasm. Protein domains, including thioredoxin, moesin tail and myosin like IQ motif domains were markedly enriched in crotonylated proteins. Kyoto Encyclopedia of Genes and Genomes and functional enrichment analyses suggested significant enrichment of crotonylated proteins in complement and coagulation cascades, and antigen processing and presentation pathways displaying important relationships with IgA nephropathy. The present results suggested that crotonylation occurred in numerous proteins and may play key regulatory roles in IgA nephropathy.

Correlation between PKB/Akt, GSK-3ß expression and tubular epithelial-mesenchymal transition in renal allografts with chronic active antibody-mediated rejection.

Chronic antibody-mediated rejection (ABMR) is a major cause of the transplant renal interstitial fibrosis and transplanted kidney epithelial cell transdifferentiation is one of the main mechanisms. The transforming growth factor (TGF)-ß1/integrin-linked kinase (ILK) signaling pathway has a significant role in the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells; however, the molecular mechanisms of this process have remained elusive. The present study confirmed that Akt and glycogen synthase kinase (GSK)-3ß, as TGF-ß1 downstream signaling factors, are involved in fibrotic processes caused by kidney disease, which, however, has been rarely reported in the kidney transplant field. Based on the Banff 2009 standard, transplanted kidney specimens were classified according to the fibrosis level. The results showed that with the reduction of the interstitial fibrosis level, E-cadherin expression was gradually reduced, while α-smooth muscle actin expression progressively increased. The expression of Akt and GSK-3ß in normal human kidney tissue was not obvious but showed a marked increase with the aggravation of the interstitial fibrosis level, which confirmed the occurrence of EMT during the fibrosis process, and that phosphorylated (p)-Akt and GSK-3ß have an important role in the EMT process in the transplanted kidney. A correlation analysis of p-Akt, GSK-3ß, TGF-ß1 and ILK suggested that overexpression of p-Akt and GSK-3ß may induce and mediate the transdifferentiation of renal tubular epithelial cells to myofibroblasts and that this proceeds via TGFß1/ILK signaling pathways.

Composition and variation analysis of the TCR ß-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing.

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRßV segments and 6 TRßJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.

Comparative proteomic analysis of renal tissue in IgA nephropathy with iTRAQ quantitative proteomics.

Immunoglobulin (Ig) A nephropathy (IgAN) is the most common form of glomerulonephritis. In clinical practice, it is difficult to monitor the repeating relapse in patients suffering from IgAN, which usually occurs within 10 years of end-stage renal disease. In order to identify and quantify the total protein content in the renal tissue of patients with IgAN, isobaric tags for relative and absolute quantification (iTRAQ) technology was performed. iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry was used to analyze the total protein of normal renal tissue in IgAN and healthy patients. The individual proteins were identified by the Mascot search engine and any that were differentially expressed were monitored. A total of 574 different proteins were identified, and 287 proteins were up- or downregulated by >1 fold alteration in levels. The results showed that iTRAQ-based quantitative proteomic technology for the identification and relative quantitation of the renal tissue proteome is efficiently applicable. The differential expression of the proteome profiles for IgAN patients was determined. Further studies using large cohorts of patient samples with long-term clinical follow-up data should be conducted to evaluate the usefulness of the pathogenesis and novel biomarker candidates of IgAN, which may develop a novel technique for the diagnosis of IgAN.

Differential proteomic analysis of renal tissue in mesangial proliferative glomerulonephritis using iTRAQ technology.

BACKGROUND: Mesangial proliferative glomerulonephritis (MePGN) is characterized by excessive mesangial cell proliferation and mesangial matrix expansion, which lead to glomerular sclerosis and obliteration and, in turn, to deteriorating renal function. To identify and quantify the total proteins in renal tissues of MePGN patients, we used isobaric tags for relative and absolute quantification (iTRAQ) technology, and then looked for differentially expressed proteome profiles in MePGN patients. METHODS: Eight-plex iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry was used to analyze total proteins in renal tissues of MePGN patients. Proteins were identified using Mascot, compared to show any differential expression. RESULTS: Among 512 distinct proteins identified, 113 proteins were up-regulated or down-regulated with a onefold or more alteration in levels across groups. Among of them, there was significant variation in our present iTRAQ study, which contains lamin A, actin, profilin-1, annexin-A1 and A2 up-regulated, and antiquitin and aldolase B down-regulated. CONCLUSION: iTRAQ-based quantitative proteomic technology is efficiently applicable for protein identification and relative quantitation of proteomes of renal tissue. Differentially expressed proteome profiles of MePGN patients are determined. Further investigation of the molecular mechanism of the involved proteins may help to better understand the pathogenesis of MePGN and to discover novel biomarker candidates, which may enable the development of new approaches to diagnosis of MePGN.

Expression of MMP-2 and TIMP-1 in renal tissue of patients with chronic active antibody-mediated renal graft rejection.

OBJECTIVE: To investigate the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metallopropteinase-1 (TIMP-1) in the renal allografts of patients with chronic active antibody-mediated rejection (AMR), and to explore their role in the pathogenesis of AMR. METHODS: Immunohistochemistry assay and computer-assisted image analysis were used to detect the expression of MMP-2 and TIMP-1 in the renal allografts with interstitial fibrosis and tubular atrophy (IF/TA) in 46 transplant recipients and 15 normal renal tissue specimens as the controls. The association of the expression level of either MMP-2 or TIMP-1 with the pathological grade of IF/TA in AMR was analyzed. RESULTS: The expression of either MMP-2 or TIMP-1 was significantly increased in the renal allografts of the recipients as compared with the normal renal tissue (P < 0.05). MMP-2 expression tended to decrease, while TIMP-1 and serum creatinine increased along with the increase of pathological grade of IF/TA (P < 0.05). In IF/TA groups, the expression of TIMP-1 was positively correlated to serum creatinine level (r = 0.718, P < 0.05). CONCLUSIONS: It is suggested by the results that abnormal expressions of MMP-2 and TIMP-1 might play roles in the development of renal fibrosis in chronic AMR. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1128474926172838.

Expression of GSK-3ß in renal allograft tissue and its significance in pathogenesis of chronic allograft dysfunction.

OBJECTIVE: To explore the expression of Glycogen synthase kinase 3 beta (GSK-3ß) in renal allograft tissue and its significance in the pathogenesis of chronic allograft dysfunction. METHODS: Renal allograft biopsy was performed in all of the renal allograft recipients with proteinuria or increased serum creatinine level who came into our hospital from January 2007 to December 2009. Among them 28 cases was diagnosed as chronic allograft dysfunction based on pahtological observation, including 21 males with a mean age of 45 ± 10 years old and 7 females with a mean age of 42 ± 9 years old. The time from kidney transplantation to biopsy were 1-9 (3.5) years. Their serum creatinine level were 206 ± 122 umol/L. Immunohistochemical assay and computer-assisted genuine color image analysis system (imagepro-plus 6.0) were used to detect the expression of GSK-3ß in the renal allografts of 28 cases of recipients with chronic allograft dysfunction. Mean area and mean integrated optical density of GSK-3ß expression were calculated. The relationship between expression level of GSK-3ß and either the grade of inflammatory cell infiltration or interstitial fibrosis/tubular atrophy in renal allograft was analyzed. Five specimens of healthy renal tissue were used as controls. RESULTS: The expression level of the GSK-3ß was significantly increased in the renal allograft tissue of recipients with chronic allograft dysfunction, compared to normal renal tissues, and GSK-3ß expression became stronger along with the increasing of the grade of either inflammatory cell infiltration or interstitial fibrosis/tubular atrophy in renal allograft tissue. CONCLUSION: There might be a positive correlation between either inflammatory cell infiltration or interstitial fibrosis/tubular atrophy and high GSK-3ß expression in renal allograft tissue. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/9924478946162998.

Differential proteomic analysis of renal tissue in lupus nephritis using iTRAQ reagent technology.

In clinical practice, it is difficult to monitor the repeating relapse in patients suffering from systemic lupus erythematosus (SLE), who usually associated with some potential complications, for example, lupus nephritis (LN), repetition renal biopsy is necessary to determine LN flares. To identify and quantify the total proteins in renal tissue of LN patients, isobaric tags for relative and absolute quantification (iTRAQ) technology was performed. Eight-plex iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyze total proteins in renal tissue of LN patients and healthy controls. Proteins were identified by mascot, which expressed differentially were noted. A total of 490 distinct proteins were identified, 113 proteins were up-regulation or down-regulation at one fold or more alteration in levels. Among of them, there was significant deviation of four proteins between our present iTRAQ study, which are up-regulated heterogeneous nuclear ribonucleoprotein (hnRNP-), Annexins and down-regulated Argininosuccinate synthetase (ASS), aldolase. iTRAQ-based quantitative proteomic technology is efficiently applicable for identification and relative quantitation of proteome of renal tissue. Differentially expressed proteome profiles of LN patients are determined. And further investigation is necessary using large cohorts of patient samples with long-term clinical follow-up data, to assess the usefulness of the pathogenesis and novel biomarker candidates of LN, which may develop a new way for diagnosis of LN.

Expression and role of integrin-linked kinase and collagen IV in human renal allografts with interstitial fibrosis and tubular atrophy.

OBJECTIVE: To investigate the expression of integrin-linked kinase (ILK) and collagen IV in renal allografts with interstitial fibrosis and tubular atrophy (IF/TA) in kidney transplant recipients, and explore its relationship with transforming growth factor-beta(1) (TGF-beta(1)) expression and the pathogenesis of IF/TA. METHODS: Immunohistochemical assay and computer-assisted genuine colored image analysis system were used to detect the expression of ILK, TGF-beta(1) and collagen IV in the renal allografts with IF/TA. The association between TGF-beta(1), collagen IV and ILK, as well as the relationship between their expressions and the pathological class of IF/TA, was analyzed. 10 specimens of healthy renal tissue were used as controls. RESULTS: The expression levels of ILK, TGF-beta(1) and collagen IV in renal allografts were significantly higher, compared to normal renal tissues (P<0.001), and the expressions tended to increase along with the increase of pathological class of IF/TA. In IF/TA group, the expression of ILK was positively correlated with the expression of TGF-beta(1) and collagen IV (r=0.976 and r=0.912, respectively; P<0.001 for both). CONCLUSION: It is suggested that by the data that ILK might mediate the mechanism through which TGF-beta(1) promote the abnormal deposition of ECM in renal allografts with IF/TA. ILK might play an important role in the progression of the interstitial fibrosis and tubular atrophy of human renal allografts and the development of chronic renal allograft dysfunction.

Clinical study of the risk factors of insulin resistance and metabolic syndrome after kidney transplantation.

OBJECTIVE: To investigate the risk factors of insulin resistance (IR) and the role of IR and metabolic syndrome in the pathogenesis of chronic allograft nephropathy (CAN). METHODS: One hundred and twenty-seven kidney transplant recipients with normal renal function and no proteinuria at the 6th month after transplantation, and without the experience of acute rejection, calcinurine intoxication and severe infection, were involved in the study. Their primary disease of ESRF was chronic glomerulonephritis but not diabetes mellitus and hypertension. Half year and one year after transplantation, blood and urine biochemical determinations and physical examination were performed in the recipients, and HOMA calculated. 200 ordinary community residents were randomized selected as controls. RESULT: The incidence of MS in the recipients was significantly higher than controls. The incidences of obesity and overweight between recipients and controls were no significant difference. While the insulin resistance level and urine albumin level, and the incidence of MS and microalbuminuria (MAU) were significantly higher in recipients with obesity or overweight than that in recipients without obesity or overweight. The insulin resistance level in tacrolimus-treated recipients was markedly higher than CsA-treated recipients, and there was a positive correlation between the blood concentration of tacrolimus and insulin resistance level. MAU positive recipients had higher insulin resistance levels than the recipients without MAU. The recipients with metabolic syndrome had higher insulin resistance levels compared to recipients without metabolic syndrome, and higher insulin resistance levels existed in recipients with hypertriglyceridemia or hypercholesterolemia, hypertension. CONCLUSION: It is shown in the study that obesity or overweight, tacrolimus (especially when its blood concentration was high) were risk factors resulting in insulin resistance in kidney transplant recipients. It is suggested in the study that insulin resistance often accompanied with hypertriglyceridemia, hypercholesterolemia and hypertension in kidney transplant recipients might be involved in the pathogenesis of the pathogenesis of CAN.