Diagnostic value of VDBP and miR-155-5p in diabetic nephropathy and the correlation with urinary microalbumin.

This study explored the diagnostic and therapeutic significance of vitamin D binding protein (VDBP) and miR-155-5p for diabetic nephropathy and the correlation with urinary microalbumin. A total of 145 patients with type 2 diabetes who attended the Hwamei hospital were selected as research objects and assigned to diabetic nephropathy group (DN group) and diabetes group according to whether they suffered from diabetic nephropathy (DN). The expression levels of urine VDBP and serum miR-155-5p in the two groups were detected, and the correlation between urinary microalbumin (mAlb), serum cystatin C (Cys C) and 24-h urinary protein was analyzed. The predictive value of single and joint detection of urinary VDBP and serum miR-155-5p for DN onset and poor prognosis was analyzed. In DN patients, urine VDBP and serum miR-155-5p were highly expressed, and urine VDBP, serum miR-155-5p and mAlb, Cys C and 24-h urine protein were positively correlated (P<0.05). Moreover, the joint detection of urine VDBP and serum miR-155-5p was more valuable in diagnosis and poor prognosis prediction of DN patients than its single detection. Urine VDBP and serum miR-155-5p have good diagnostic value for DN, but their joint diagnostic value is higher, and their expression levels are all related to mAlb of DN patients, which may be used as new biological indicators for diagnosis and disease assessment.

Dihydromyricetin promotes autophagy and attenuates renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling in diabetic nephropathy.

Diabetic nephropathy (DN) is the most common complication of diabetes and is prone to kidney failure. Dihydromyricetin (DHM) has been reported to have a variety of pharmacological activities. This study aims to explore the effect of DHM on DN and the underlying molecular mechanism. An in vivo DN rat model was established. The degree of renal interstitial fibrosis (RIF) was detected by hematoxylin-eosin (HE) staining, Masson's trichrome staining, and immunohistochemistry (IHC). In vitro, NRK-52E cells were divided into four groups: normal glucose (NG), high glucose (HG), HG+DHM, and HG+rapamycin (autophagy inhibitor). The levels of autophagy- and fibrosis-related proteins were analyzed by western blotting. The expression of miR-155-5p and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and their relationship were assessed by quantitative reverse transcription (qRT)-PCR and dual luciferase reporter gene assay. Our results showed that RIF was increased in DN rat model and in HG-induced NRK-52E cells. DHM treatment attenuated the increased RIF and also increased autophagy. MiR-155-5p expression was increased, while PTEN expression was decreased in DN rat and cell model, and DHM reversed both effects. Dual luciferase assay showed that PTEN was the target gene of miR-155-5p. DHM inhibited HG-induced fibrosis and promoted autophagy by inhibiting miR-155-5p expression in NRK-52E cells. In addition, DHM promoted autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway. In conclusion, DHM promotes autophagy and attenuates RIF by regulating the miR-155-5p/PTEN signaling and PI3K/AKT/mTOR signaling pathway in DN.

Microarray analysis of long non-coding RNA expression in human acute rejection biopsy samples following renal transplantation.

Rejection is still a major obstacle in long-term allograft survival of renal transplant recipients. Long non­coding RNAs (lncRNAs) are an important class of pervasive RNAs involved in a variety of biological functions, and which are often found to be differentially expressed between healthy and pathological conditions. The aim of this study was to compare the expression profiles of lncRNAs between samples from acute rejection following kidney transplantation and control samples. Three patients were enrolled, diagnosed by renal biopsy with acute rejection upon kidney transplantation. We used lncRNA microarrays to study the lncRNA expression profiles in the kidney biopsies of these patients and in kidneys from healthy donors. Reverse transcription­quantitative polymerase chain reaction (RT-qPCR) was used to validate the microarray results. In addition, potential functions of the identified lncRNAs were further explored by searching the UCSC, RNAdb, RefSeq and NRED databases. Five candidate lncRNAs displaying differential expression in acute rejection samples were validated by RT-qPCR. The results were in agreement with the microarray data. Among the identified lncRNAs, certain have been previously identified in relevant conditions, thereby supporting previous evidence, but certain may constitute novel biomarker candidates. This is the first report to date using lncRNA microarrays to identify unique expression signatures of acute rejection in transplant biopsies. Our data indicate that lncRNAs are potentially involved in the pathogenesis of acute rejection. Our results may have important implications in the identification of diagnostic biomarkers, as well as in the understanding and treatment of acute rejection following renal transplantation.

Analysis of microRNAs in patients with systemic lupus erythematosus, using Solexa deep sequencing.

OBJECTIVES: Our aim in this study was to identify and examine the differential expression of microRNAs in patients with systemic lupus erythematosus (SLE). METHODS: We employed high-quality, high-throughput Solexa sequencing to clone and identify microRNAs in SLE patients and a control group. RESULTS: From the sequencing data, we identified numerous microRNAs displaying significantly different levels of expression in patients with SLE and in healthy controls. The 212 and 199 microRNAs were upregulated and downregulated, respectively. Only 61 novel microRNAs exhibited significantly different levels of expression in the two groups. The target genes of the novel microRNAs identified in the SLE group were found to have cell metabolism functions. We also analyzed the chromosomal locations of the microRNAs with high level of expression between the two groups. A profile comparison revealed that the majority of transcripts were expressed at a similar level. The functional classes of the most abundant microRNAs were equally represented on each chromosome. CONCLUSION: We identified novel and known microRNAs significantly enhancing our understanding of the microRNA expression profiles of SLE patients. These data also provide insight into the function of microRNAs in SLE and provide new strategies for future therapies.

Integrated profiling of microRNA expression in membranous nephropathy using high-throughput sequencing technology.

The present study analyzed microRNA (miRNA) expression profiles in peripheral blood lymphocyte cells (PBLCs) from patients with membranous nephropathy (MN) and normal controls (NC), in an effort to improve the understanding of the pathogenesis of MN. High-throughput sequencing was performed on 30 MN patients and 30 healthy individuals (NC group). Known and novel miRNAs were analyzed and the results were confirmed by quantitative reverse transcription PCR (qRT-PCR). In total, 326 miRNAs showed a significant difference in expression between the MN and NC groups. This included 286 downregulated miRNAs and 40 upregulated miRNAs. In addition, there were 6 novel miRNAs that presented differential levels of expression between the MN and NC groups. The miRNAs were mapped to the genome, using a short oligonucleotide alignment program (SOAP), to analyze their expression and distribution. Twenty-five percent of the unique miRNAs in the MN group and 52.1% in the NC group were mapped to the genome. One hundred and eight mismatches were identified. Seventy-seven mismatches were detected in a higher proportion of the MN samples, compared with the NC samples. Twenty-five mismatches were detected in a higher proportion of the NC samples than the MN samples. Differential miRNA expression was also detected between 10 randomly selected pair groups, as depicted in a cluster analysis diagram. These data indicate that differential miRNA expression may be involved in the pathogenesis of MN. In addition, the discrepancies between the MN and NC groups, in the mismatched miRNAs that were mapped to the genome, strongly suggest that miRNAs play an important role in the pathogenesis of human disorders. miRNAs may provide a potential breakthrough in the research of MN and may provide a novel biomarker for the diagnosis and treatment of the disease.

Identification of dysregulated microRNAs in lymphocytes from children with Down syndrome.

Given the important roles of miRNAs in post-transcriptional regulation and its implications for the development of immune tissues and cells, characterization of miRNAs promotes us to uncover the molecular mechanisms underlying the pathway of trisomic chromosome 21 that disrupts the disomic genes expression and immunological defects related to Down syndrome (DS). In the present study, we analyzed global changes and chromosome distribution characteristics of miRNAs expression in lymphocytes from children with trisomy 21 by means of the Illumina high-throughput sequencing technology. Two small libraries were constructed using pool RNA of normal and DS children. The results have been further validated by stem-loop quantitative RT-PCR. Comparison between DS and normal profiles revealed that most of identified miRNAs were expressed at similar levels. The chromosome 21 that contributes to the abundantly expressed miRNAs was small, and not all Hsa21-derived miRNAs were over-expressed with ratios significantly ≥ 1.5 in Down syndrome children lymphocytes. Based on the deep sequencing technology, 108 novel candidate miRNAs have been identified, and 2 of them were derived from human chromosome 21. For the 114 significantly differentially expressed miRNAs, function annotation of target genes indicated that a set of highly abundantly and significantly differentially expressed miRNAs were involved in hematopoietic or lymphoid organ development, thymus development, and T/B cell differentiation and activation. Our results indicated that these abnormally expressed miRNAs might be associated with the mechanisms that trisomy 21 results in dysregulation of disomic genes and involved in the immunological defects seen in DS.

Genome-wide analysis of microRNAs expression profiling in patients with primary IgA nephropathy.

The aim of this study was to investigate the differential expression characteristics and the roles of the genome-wide microRNAs (miRNAs) in immunoglobulin A nephropathy (IgAN) kidney tissues. We used Illumina high-throughput sequencing technology to evaluate the miRNAs expression of six biopsy tissues from IgAN and six normal renal cortex specimens from patients with renal cell carcinoma. We observed a total of 85 miRNAs that were differentially expressed in the six IgAN patients, of which 11 miRNAs were up-regulated and 74 miRNAs were down-regulated in patients' tissues compared with control tissues. Additionally, we identified 55 candidate novel miRNAs in our study, which comprised seven candidates who were detected in the IgAN group and 49 candidates who were detected in the control group. Only one candidate (miR-n-9) was expressed in both groups. The bioinformatics showed that the regulated target genes of differentially expressed miRNAs were associated with immune and renal pathological changes. The identification of specific tissue miRNAs in our study not only helped clarify the genetics or immunology mechanisms involved in the pathogenesis of IgAN but also helped explain the pathological changes in the kidney tissues. We hypothesize that some significant miRNAs might potentially serve as novel diagnostic biomarkers in IgAN patients.

Generation of systemic lupus erythematosus-specific induced pluripotent stem cells from urine.

Systemic lupus erythematosus (SLE) is the prototype of complex autoimmune diseases characterized by the production of autoantibodies which results in widespread immunologic abnormalities and immune complex formation. The underlying etiology remains largely unknown. When progressing toward kidney failure, it is becoming a serious public health problem. Kidney transplantation is a feasible therapy, but significant limitations were existed, including shortage of donor organs and lack of funding. To find an alternative proposal for kidney replacement, the induced pluripotent stem cells (iPSCs) technology was adopted. We identified typical SLE patients. Lentiviral transduction of OCT4, SOX2, KLF4, and c-MYC, under feeder conditions, resulted in reprogramming of urine-derived renal tubular cells. We investigated the viability of iPSCs generation from patients with SLE by identification of totipotency and pluripotency. SLE patient renal tubular cells-derived iPSCs exhibited properties of human embryonic stem cells, including morphology, growth properties, alkaline phosphatase, expression of pluripotency, genes and surface markers, and teratoma formation. We demonstrated that generation of SLE-specific iPSCs from urine was not only the first time worldwide, but was feasible and efficient. IPSCs from SLE would provide convenient model to study disease pathogenesis, drugs screening, and gene therapy.