Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial‐to‐mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor‐beta1 (TGF‐β1) at different doses or at different intervals with western blotting, qRT‐PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF‐β1 was determined by western blotting analysis of factors involved in various canonical and non‐canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF‐β1 significantly promoted the development of EndMT in a time‐dependent and dose‐dependent manner and promoted the motility and migration ability of HUVECs. The TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF‐β1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients.
Background: The modification and regulation of N6-methyladenosine (m 6 A) at mRNA level can affect the development and progression in various tumors. ALKBH5, as an m 6 A demethylase, plays different roles in tumors by regulating the m 6 A modification of mRNA. However, its role in renal cell carcinoma (RCC) remains unclear.Methods: First, levels of ALKBH5 in RCC tissues and cell lines were verified by qRT-PCR and western blot. We analyzed the relationship between ALKBH5 and the clinicopathological characteristics of RCC patients and the influence of ALKBH5 on the prognosis of patients. Then we generated ALBKH5overexpression, ALBKH5-knockdown stable RCC cell lines and their control cell lines. Through cell proliferation assay, colony formation assay, cell invasion and tumor migration assay, cell cycle assay and xenograft studies, we studied the ALKBH5 roles in RCC cell lines. AURKB was predicted to be its potential target based on TCGA database analysis and verified by western blot. The role of AURKB in RCC was verified by TCGA database and Kaplan-Meier analysis with TMA immunohistochemical analysis. Finally, the specific molecular mechanism of ALKBH5 targeting AURKB was explored by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), m 6 A dot-blot assay, m 6 A RNA Immunoprecipitation (MeRIP) assay, and mRNA stability assay.Results: We found that ALKBH5 was highly expressed in both RCC tumor tissues and cell lines.Clinicopathological analysis showed that high ALKBH5 expression was associated with larger tumor volume (P=0.017) and higher TNM staging (P=0.006), and worse prognosis (log rank: P=0.0199). The cellular functional assays showed that stably overexpression ALKBH5 could promote the cell proliferation, colony formation, cell migration and cell invasion of renal cell carcinoma cells in vitro and promote tumor growth in vivo. In contrast, ALKBH5 knocked down inhibited cell proliferation, colony formation, migration and invasion of renal cell carcinoma cells in vitro. Based on TCGA database analysis, AURKB was predicted highly expressed in RCC and a potential target of ALKBH5. Both database prediction and TMA immunohistochemical analysis supported that AURKB could affect the prognosis of RCC patients (P values of 5.5e-08 and 0.0004, respectively) and was regulated by ALKBH5 expression level. Subsequent mechanism experiments showed that ALKBH5 regulated the expression of AURKB by regulating the stability of AURKB mRNA in the m 6 A-dependent manner, and finally promoted cell proliferation. Furthermore, we found that hypoxia-induced HIF could up-regulate both expressions of AURKB and ALKBH5.Conclusions: Our findings suggest that ALKBH5 may play a carcinogenic role in renal cell carcinoma by stabilizing AURKB mRNA in a m 6 A-dependent manner. These data suggest that ALKBH5 may play a key role in RCC and targeting the ALKBH5 signaling pathway may be a promising strategy for the treatment of RCC.
Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.
BackgroundWe examined the usefulness of the nuclear matrix protein 22 (NMP22) BladderChek test for detecting bladder cancer.Materials and MethodsA literature search was performed using PubMed, Embase, the Cochrane Library, and Web of Science. The diagnostic accuracy of the NMP22 BladderChek test was evaluated via pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under curve (AUC). Inter-study heterogeneity was explored using meta-regression and subgroup analyses.ResultsWe included 23 studies in the systematic review and 19 in the quantitative meta-analysis. Overall sensitivity and specificity were 56% (52–59%) and 88% (87–89%), respectively; pooled PLR and NLR were 4.36 (3.02–6.29) and 0.51 (0.40–0.66), respectively; DOR was 9.29 (5.55–15.55) with an AUC of 0.8295. The mean sensitivity for Ta, T1, ≥ T2, Tis, G1, G2, and G3 disease was 13.68%, 29.49%, 74.03%, 34.62%, 44.16%, 56.25%, and 67.34%, respectively.ConclusionsThe NMP22 BladderChek test shows good discrimination ability for detecting bladder cancer and a high-specificity algorithm that can be used for early detection to rule out patients with higher bladder cancer risk. It also has better potential for screening higher-grade and higher-stage tumors, and better diagnostic performance in Asians.
Nuclear factor-κB is associated with the pathogenesis of numerous malignancies, and the functional polymorphism −94ins/del ATTG (rs28362491) in the human NFKB1 gene is associated with cancer risk. Previous studies on the association between the −94ins/del ATTG polymorphism and cancer risk reported conflicting results. To clarify this relationship, we performed a meta-analysis of 21 case-control studies involving 6127 cases and 9238 controls. We used pooled odds ratios (ORs) with their 95% confidence intervals (95% CIs) to assess the association. We found that the NFKB1 promoter −94ins/del ATTG polymorphism was significantly associated with cancer risk in four genetic models (ins/ins versus del/del, OR = 1.47, 95% CI = 1.11–1.93; dominant model, OR = 1.26, 95% CI = 1.03–1.53; recessive model, OR = 1.26, 95% CI = 1.05–1.51; ins allele versus del allele, OR = 1.19, 95% CI = 1.05–1.35). Stratified analyses revealed a significant association between the polymorphism and ovarian, oral, and prostate cancers. Similar results were determined in an Asian population and not in a Caucasian population. Thus, our results suggested that the polymorphism can contribute to cancer risk. Moreover, the polymorphism can exert race- and cancer-specific effects on cancer risk. Further large-scale and functional studies are necessary to elucidate this possible effect.
Background RNA-seq is poised to play a major role in the management of kidney transplant patients. Rigorous definition of housekeeping genes (HKG) is essential for further progress in this field. Using single genes or a limited set HKG is inherently problematic since their expression might be altered by specific diseases in the patients being studied. Methods To generate a HKG set specific for kidney transplantation, we performed RNA-sequencing from renal allograft biopsies collected in a variety of clinical settings. Various normalization methods were applied to identify transcripts that had a coefficient of variation of expression that was below the 2nd percentile across all samples, and the corresponding genes were designated as housekeeping genes. Comparison with transcriptomic data from the Gene Expression Omnibus (GEO) database, pathway analysis and molecular biological functions were utilized to validate the housekeeping genes set. Results We have developed a bioinformatics solution to this problem by using nine different normalization methods to derive large HKG gene sets from a RNA-seq data set of 47,611 transcripts derived from 30 biopsies. These biopsies were collected in a variety of clinical settings, including normal function, acute rejection, interstitial nephritis, interstitial fibrosis/tubular atrophy and polyomavirus nephropathy. Transcripts with coefficient of variation below the 2nd percentile were designated as HKG, and validated by showing their virtual absence in diseased allograft derived transcriptomic data sets available in the GEO. Pathway analysis indicated a role for these genes in maintenance of cell morphology, pyrimidine metabolism, and intracellular protein signaling. Conclusions Utilization of these objectively defined HKG data sets will guard against errors resulting from focusing on individual genes like 18S RNA, actin & tubulin, which do not maintain constant expression across the known spectrum of renal allograft pathology.
Alzheimer's disease is the leading cause of dementia. The long progression period in Alzheimer's disease provides a possibility for patients to get early treatment by having routine screenings. However, current clinical diagnostic imaging tools do not meet the specific requirements for screening procedures due to high cost and limited availability. In this work, we took the initiative to evaluate the retina, especially the retinal vasculature, as an alternative for conducting screenings for dementia patients caused by Alzheimer's disease. Highly modular machine learning techniques were employed throughout the whole pipeline. Utilizing data from the UK Biobank, the pipeline achieved an average classification accuracy of 82.44%. Besides the high classification accuracy, we also added a saliency analysis to strengthen this pipeline's interpretability. The saliency analysis indicated that within retinal images, small vessels carry more information for diagnosing Alzheimer's diseases, which aligns with related studies.
The aim was to investigate the clinical efficacy and safety of extracorporeal shock wave lithotripsy (ESWL) in pediatric urolithiasis. A comprehensive systematic review and meta-analysis were performed. PubMed, Embase, and the Cochrane central register of controlled trials (CENTRAL) were searched, and the stone-free rates (SFRs) of various stone sizes and stone positions were extracted from the eligible articles. The quality of the original articles was assessed according to the McHarm Scale. The risk ratios (RRs) and 95% confidential intervals (CIs) were pooled, and the sensitive analysis was performed to evaluate the heterogeneity among all eligible studies. In total, 14 studies with 1842 patients were identified. The pooled RR for the SFR of stones less than 10 mm and greater than 10 mm was 1.14 (95% CI: 1.07, 1.21, P < 0.001); the RR for the SFR of stones in the renal pole calix (PC) and the renal pelvis was 0.95 (95% CI: 0.893, 1.009, P < 0.01); the RR for the SFR of stones in the upper/middle PC and the lower PC was 1.07 (95% CI: 0.997, 1.156, P < 0.061); and the RR for the SFR of stones in the proximal ureter and middle/distal ureter was 1.077 (95% CI: 1.005, 1.154, P = 0.036). Heterogeneity was low in all the analyses. Major complications in ESWL of pediatric urolithiasis were steinstrasse and abdominal colic, the incidences of which were 6.00 and 6.29 %, respectively. The SFR of stones <10 mm was significantly higher than stones >10 mm, and the SFR of stones located in proximal ureter was statistically greater than stones in middle or distal ureter in pediatric urolithiasis, leaving no significant between stones in renal PC and renal pelvis, or between upper/middle PC and lower PC. The use of ESWL in children is highly efficient, with negligible complications; ESWL therapy could be considered the first-line treatment for pediatric urolithiasis.
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