Identification of key genes and pathways in diabetic nephropathy by bioinformatics analysis.

AIMS/INTRODUCTION: The aim of the present study was to identify candidate differentially expressed genes (DEGs) and pathways using bioinformatics analysis, and to improve our understanding of the cause and potential molecular events of diabetic nephropathy. MATERIALS AND METHODS: Two cohort profile datasets (GSE30528 and GSE33744) were integrated and used for deep analysis. We sorted DEGs and analyzed differential pathway enrichment. DEG-associated ingenuity pathway analysis was carried out. The screened gene expression feature was verified in the db/db mouse kidney cortex. Then, rat mesangial cells cultured with high-concentration glucose were used for verification. The target genes of transcriptional factor E26 transformation-specific-1 (ETS1) were predicted with online tools and validated using chromatin immunoprecipitation assay quantitative polymerase chain reaction. RESULTS: The two GSE datasets identified 89 shared DEGs; 51 were upregulated; and 38 were downregulated. Most of the DEGs were significantly enriched in cell adhesion, the plasma membrane, the extracellular matrix and the extracellular region. Quantitative reverse transcription polymerase chain reaction analysis validated the upregulated expression of Itgb2, Cd44, Sell, Fn1, Tgfbi and Il7r, and the downregulated expression of Igfbp2 and Cd55 in the db/db mouse kidney cortex. Chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that Itgb2 was the target gene of transcription factor Ets1. ETS1 knockdown in rat mesangial cells decreased integrin subunit beta 2 expression. CONCLUSION: We found that EST1 functioned as an important transcription factor in diabetic nephropathy development through the promotion of integrin subunit beta 2 expression. EST1 might be a drug target for diabetic nephropathy treatment.

New Mutation of Coenzyme Q10 Monooxygenase 6 Causing Podocyte Injury in a Focal Segmental Glomerulosclerosis Patient.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function, which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments. The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis. Here, we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. METHODS: Using next-generation sequencing and Sanger sequencing, we screened the causative gene that was linked to FSGS in this study. The patient's total blood RNA was extracted to validate the messenger RNA (mRNA) expression of coenzyme Q10 monooxygenase 6 (COQ6) and validated it by immunohistochemistry. COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then, F-actin was determined using immunofluorescence staining. Cell apoptosis was evaluated by flow cytometry, the expression of active caspase-3 was determined by Western blot, and mitochondrial function was detected by MitoSOX. RESULTS: Using whole-exome sequencing and Sanger sequencing, we screened a new causative gene, COQ6, NM_182480: exon1: c.G41A: p.W14X. The mRNA expression of COQ6 in the proband showed decreased. Moreover, the expression of COQ6, which was validated by immunohistochemistry, also had the same change in the proband. Finally, we focused on the COQ6 gene to clarify the mechanism of podocyte injury. Flow cytometry showed significantly increased in apoptotic podocytes, and Western blotting showed increases in active caspase-3 in si-COQ6 podocytes. Meanwhile, reactive oxygen species (ROS) levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. CONCLUSIONS: This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6, which could cause respiratory chain defect, increase the generation of ROS, destroy the podocyte cytoskeleton, and induce apoptosis. It provides basic theoretical basis for the screening of FSGS in the future.

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) has become a public health problem. New interventions to slow or prevent disease progression are urgently needed. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of embryonic stem cells (ESCs) on the progression of CKD. METHODS: Adult male Sprague-Dawley rats were subjected to 5/6 nephrectomy. We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogels (GMs) on the 5/6 nephrectomized kidney. The viability of ESCs within the GMs was detected using in vitro two-photon fluorescence confocal imaging. Rats were sacrificed after 12 weeks. Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results. Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining. RESULTS: In vitro, ESCs could be automatically loaded into the GMs. Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro. After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs. CONCLUSIONS: In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury.

[Analysis on children timely vaccination coverage and influencing factors of 5 kinds of expanded program for immunization (EPI) vaccine in Dinghai District of Zhoushan Municipal].

OBJECTIVE: To understand the timely coverage of 5 kinds of EPI vaccine in Dinghai District, including hepatitis B vaccine (HepB), bacillus calmette guerin vaccine (BCG), oral poliomyelitis attenuated live vaccine (OPV), diphtheria-pertussis-tetanus combined vaccine (DPT), measles attenuated live vaccine (MV), and to analyze the influence factors so as to formulate measures to improve the coverage. METHODS: Analyzing the immunization data and related information of 4258 resident children and 394 floating population born during 2005-2007. RESULTS: Timely coverage rate of BCG, HepB, OPV, DPT, MV were 22.26%, 95.020, 90.82%, 91.40%, 95.40% respectively and 48.73%, 74.37%, 76.40%, 80.46%, 84.52%, for floating population. Except for the timely BCG coverage rate was low for resident and floating populations, the timely vaccination rates for other 4 kinds of NIP vaccines in the resident were significantly higher than those in the floating population. The logistic regression analysis showed that inappropriate timing of vaccination, long time waiting for inoculation, lacking of time for the families, long distance from the vaccination sites were four factors that affected the timely immunization of NIP vaccines. CONCLUSION: Implementing the strategy of midwife being responsible for BCG and HepB vaccination can improve the timely coverage of the first dose HepB and BCG: Shorten running inteval of vaccination and available service from the supplier, improve immunization awareness and initiative of the recipients (especially for the floating children's parent/guardian) will be useful to increase the timely NIP vaccine coverage rates.