Human umbilical cord mesenchymal stem cell exosomes alleviate acute kidney injury by inhibiting pyroptosis in rats and NRK-52E cells.

Human umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia-reperfusion injury (IRI) group, and IRI + hucMSC-Ex group. In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 µg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1ß) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis.

Human umbilical cord mesenchymal stem cell exosome-derived miR-874-3p targeting RIPK1/PGAM5 attenuates kidney tubular epithelial cell damage.

BACKGROUND: Kidney insults due to various pathogenic factors, such as trauma, infection, and inflammation, can cause tubular epithelial cell injury and death, leading to acute kidney injury and the transformation of acute kidney injury to chronic kidney disease. There is no definitive treatment available. In previous studies, human umbilical cord mesenchymal stem cells have been shown to promote kidney injury. In this preclinical study, we investigate the role and mechanism of human umbilical cord mesenchymal stem cell exosomes (HucMSC-Exos) on the repair of renal tubular epithelial cells after injury. METHODS: C57BL/6 mice underwent unilateral ureteral obstruction, and epithelial cell injury was induced in HK-2 cells by cisplatin. HucMSC-Exos were assessed in vivo and in vitro. The extent of renal cell injury, activation of necroptosis pathway, and mitochondrial quality-control-related factors were determined in different groups. We also analyzed the possible regulatory effector molecules in HucMSC-Exos by transcriptomics. RESULTS: HucMSC-Exo inhibited necroptosis after renal tubular epithelial cell injury and promoted the dephosphorylation of the S637 site of the Drp1 gene by reducing the expression of PGAM5. This subsequently inhibited mitochondrial fission and maintained mitochondrial functional homeostasis, mitigating renal injury and promoting repair. In addition, HucMSC-Exo displayed a regulatory role by targeting RIPK1 through miR-874-3p. CONCLUSION: The collective findings of the present study demonstrate that HucMSC-Exos can regulate necroptosis through miR-874-3p to attenuate renal tubular epithelial cell injury and enhance repair, providing new therapeutic modalities and ideas for the treatment of AKI and the process of AKI to CKD transformation to mitigate renal damage.

Additively manufactured controlled porous orthopedic joint replacement designs to reduce bone stress shielding: a systematic review.

BACKGROUND: Total joint replacements are an established treatment for patients suffering from reduced mobility and pain due to severe joint damage. Aseptic loosening due to stress shielding is currently one of the main reasons for revision surgery. As this phenomenon is related to a mismatch in mechanical properties between implant and bone, stiffness reduction of implants has been of major interest in new implant designs. Facilitated by modern additive manufacturing technologies, the introduction of porosity into implant materials has been shown to enable significant stiffness reduction; however, whether these devices mitigate stress-shielding associated complications or device failure remains poorly understood. METHODS: In this systematic review, a broad literature search was conducted in six databases (Scopus, Web of Science, Medline, Embase, Compendex, and Inspec) aiming to identify current design approaches to target stress shielding through controlled porous structures. The search keywords included 'lattice,' 'implant,' 'additive manufacturing,' and 'stress shielding.' RESULTS: After the screening of 2530 articles, a total of 46 studies were included in this review. Studies focusing on hip, knee, and shoulder replacements were found. Three porous design strategies were identified, specifically uniform, graded, and optimized designs. The latter included personalized design approaches targeting stress shielding based on patient-specific data. All studies reported a reduction of stress shielding achieved by the presented design. CONCLUSION: Not all studies used quantitative measures to describe the improvements, and the main stress shielding measures chosen varied between studies. However, due to the nature of the optimization approaches, optimized designs were found to be the most promising. Besides the stiffness reduction, other factors such as mechanical strength can be considered in the design on a patient-specific level. While it was found that controlled porous designs are overall promising to reduce stress shielding, further research and clinical evidence are needed to determine the most superior design approach for total joint replacement implants.

Hypoxia-Induced HIF-1α Expression Promotes Neurogenic Bladder Fibrosis via EMT and Pyroptosis.

BACKGROUND: Neurogenic bladder (NB) patients exhibit varying degrees of bladder fibrosis, and the thickening and hardening of the bladder wall induced by fibrosis will further affect bladder function and cause renal failure. Our study aimed to investigate the mechanism of bladder fibrosis caused by a spinal cord injury (SCI). METHODS: NB rat models were created by cutting the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves. RNA-seq, Western blotting, immunofluorescence, cell viability and ELISA were performed to assess the inflammation and fibrosis levels. RESULTS: The rats showed bladder dysfunction, upper urinary tract damage and bladder fibrosis after SCI. RNA-seq results indicated that hypoxia, EMT and pyroptosis might be involved in bladder fibrosis induced by SCI. Subsequent Western blot, ELISA and cell viability assays and immunofluorescence of bladder tissue confirmed the RNA-seq findings. Hypoxic exposure increased the expression of HIF-1α and induced EMT and pyroptosis in bladder epithelial cells. Furthermore, HIF-1α knockdown rescued hypoxia-induced pyroptosis, EMT and fibrosis. CONCLUSION: EMT and pyroptosis were involved in the development of SCI-induced bladder fibrosis via the HIF-1α pathway. Inhibition of the HIF-1α pathway may serve as a potential target to alleviate bladder fibrosis caused by SCI.

MK-2206 Alleviates Renal Fibrosis by Suppressing the Akt/mTOR Signaling Pathway In Vivo and In Vitro.

Renal fibrosis is a common pathological feature of various kidney diseases, leading to irreversible renal failure and end-stage renal disease. However, there are still no effective treatments to reverse renal fibrosis. This study aimed to explore the potential mechanism of a targeted drug for fibrosis. Here, unilateral ureteral obstruction (UUO)-treated mice and a TGF-ß1-treated human renal tubular epithelial cell line (HK-2 cells) were used as models of renal fibrosis. Based on the changes of mRNA in UUO kidneys detected by transcriptome sequencing, MK-2206, an Akt inhibitor, was predicted as a potential drug to alleviate renal fibrosis through bioinformatics. We dissolved UUO mice with MK-2206 by gastric gavage and cultured TGF-ß-induced HK-2 cells with MK-2206. Histopathological examinations were performed after MK-2206 intervention, and the degree of renal fibrosis, as well as the expression of Akt/mTOR pathway-related proteins, were evaluated by immunohistochemical staining, immunofluorescence staining, and Western blot. The results showed that MK-2206 significantly improved the pathological structure of the kidney. Furthermore, MK-2206 intervention effectively inhibited UUO- and TGF-ß1-induced epithelial-mesenchymal transition, fibroblast activation, and extracellular matrix deposition. Mechanistically, MK-2206 treatment attenuated the activation of the Akt/mTOR signaling pathway. Taken together, our study revealed for the first time that MK-2206 is a promising drug for the improvement of renal fibrosis.

A novel cuproptosis-related subtypes and gene signature associates with immunophenotype and predicts prognosis accurately in neuroblastoma.

Background: Neuroblastoma (NB) is the most frequent solid tumor in pediatrics, which accounts for roughly 15% of cancer-related mortality in children. NB exhibited genetic, morphologic, and clinical heterogeneity, which limited the efficacy of available therapeutic approaches. Recently, a new term 'cuproptosis' has been used to denote a unique biological process triggered by the action of copper. In this instance, selectively inducing copper death is likely to successfully overcome the limitations of conventional anticancer drugs. However, there is still a gap regarding the role of cuproptosis in cancer, especially in pediatric neuroblastoma. Methods: We characterized the specific expression of cuproptosis-related genes (CRGs) in NB samples based on publicly available mRNA expression profile data. Consensus clustering and Lasso-Cox regression analysis were applied for CRGs in three independent cohorts. ESTIMATE and Xcell algorithm was utilized to visualize TME score and immune cell subpopulations' relative abundances. Tumor Immune Dysfunction and Exclusion (TIDE) score was used to predict tumor response to immune checkpoint inhibitors. To decipher the underlying mechanism, GSVA was applied to explore enriched pathways associated with cuproptosis signature and Connectivity map (CMap) analysis for drug exploration. Finally, qPCR verified the expression levels of risk-genes in NB cell lines. In addition, PDHA1 was screened and further validated by immunofluorescence in human clinical samples and loss-of-function assays. Results: We initially classified NB patients according to CRGs and identified two cuproptosis-related subtypes that were associated with prognosis and immunophenotype. After this, a cuproptosis-related prognostic model was constructed and validated by LASSO regression in three independent cohorts. This model can accurately predict prognosis, immune infiltration, and immunotherapy responses. These genes also showed differential expression in various characteristic groups of all three datasets and NB cell lines. Loss-of-function experiments indicated that PDHA1 silencing significantly suppressed the proliferation, migration, and invasion, in turn, promoted cell cycle arrest at the S phase and apoptosis of NB cells. Conclusions: Taken together, this study may shed light on new research areas for NB patients from the cuproptosis perspective.

The Regulatory Network and Role of the circRNA-miRNA-mRNA ceRNA Network in the Progression and the Immune Response of Wilms Tumor Based on RNA-Seq.

Circular RNA (circRNA), which is a newly discovered non-coding RNA, has been documented to play important roles in miRNA sponges, and the dysregulation of which is involved in cancer development. However, circRNA expression profiles and their role in initiation and progression of Wilms tumor (WT) remain largely unclear at present. Here, we used paired WT samples and high-throughput RNA sequencing to identify differentially expressed circRNAs (DE-circRs) and mRNAs (DE-mRs). A total of 314 DE-circRs and 1612 DE-mRs were identified. The expression of a subset of differentially expressed genes was validated by qRT-PCR. A complete circRNA-miRNA-mRNA network was then constructed based on the common miRNA targets of DE-circRs and DE-mRs identified by miRanda prediction tool. The Gene set enrichment analysis (GSEA) indicated that several signaling pathways involving targeted DE-mRs within the ceRNA network were associated with cell cycle and immune response, which implies their participation in WT development to some extent. Subsequently, these targeted DE-mRs were subjected to implement PPI analysis and to identify 10 hub genes. Four hub genes were closely related to the survival of WT patients. We then filtered prognosis-related hub genes by Cox regression and least absolute shrinkage and selection operator (LASSO) regression analysis to construct a prognosis-related risk score system based on a three-gene signature, which showed good discrimination and predictive ability for WT patient survival. Additionally, we analyzed the mutational landscape of these genes and the associations between their expression levels and those of immune checkpoint molecules and further demonstrated their potential impact on the efficacy of immunotherapy. qRT-PCR and western blotting (WB) analysis were used to validate key differentially expressed molecules at the RNA and protein levels, respectively. Besides these, we selected a key circRNA, circEYA1, for function validation. Overall, the current study presents the full-scale expression profiles of circRNAs and the circRNA-related ceRNA network in WT for the first time, deepening our understanding of the roles and downstream regulatory mechanisms of circRNAs in WT development and progression. We further constructed a useful immune-related prognostic signature, which could improve clinical outcome prediction and guide individualized treatment.

Influence of the geometric and material properties of lumbar endplate on lumbar interbody fusion failure: a systematic review.

BACKGROUND: Lumbar interbody fusion (LIF) is an established surgical intervention for patients with leg and back pain secondary to disc herniation or degeneration. Interbody fusion involves removal of the herniated or degenerated disc and insertion of interbody devices with bone grafts into the remaining cavity. Extensive research has been conducted on operative complications such as a failure of fusion or non-union of the vertebral bodies. Multiple factors including surgical, implant, and patient factors influencing the rate of complications have been identified. Patient factors include age, sex, osteoporosis, and patient anatomy. Complications can also be influenced by the interbody cage design. The geometry of the bony endplates as well as their corresponding material properties guides the design of interbody cages, which vary considerably across patients with spinal disorders. However, studies on the effects of such variations on the rate of complications are limited. Therefore, this study aimed to perform a systematic review of lumbar endplate geometry and material property factors in LIF failure. METHODS: Search keywords included 'factor/cause for spinal fusion failure/cage subsidence/cage migration/non-union', 'lumbar', and 'interbody' in electronic databases PubMed and Scopus with no limits on year of publication. RESULTS: In total, 1341 articles were reviewed, and 29 articles were deemed suitable for inclusion. Adverse events after LIF, such as cage subsidence, cage migration, and non-union, resulted in fusion failure; hence, risk factors for adverse events after LIF, notably those associated with lumbar endplate geometry and material properties, were also associated with fusion failure. Those risk factors were associated with shape, concavity, bone mineral density and stiffness of endplate, segmental disc angle, and intervertebral disc height. CONCLUSIONS: This review demonstrated that decreased contact areas between the cage and endplate, thin and weak bony endplate as well as spinal diseases such as spondylolisthesis and osteoporosis are important causes of adverse events after LIF. These findings will facilitate the selection and design of LIF cages, including customised implants based on patient endplate properties.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 Are Involved in Ritonavir-Induced Cytotoxicity in Hepatoma Cells.

Ritonavir (RTV), a pharmacoenhancer used in anti-HIV regimens, can induce liver damage. RTV is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A-AS1) and HNF1A antisense RNA 1 (HNF1A-AS1) are long noncoding RNAs that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and underlying mechanisms of HNF4A-AS1 and HNF1A-AS1 in RTV-induced hepatotoxicity. HNF4A-AS1 and HNF1A-AS1 were knocked down by small hairpin RNAs in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV-induced hepatotoxicity. Chromatin immunoprecipitation quantitative real-time polymerase chain reaction was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A-AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV-induced cytotoxicity, whereas HNF1A-AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A-AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A-AS1 knockdown. Alterations in RTV-induced hepatotoxicity caused by decreasing HNF4A-AS1 or HNF1A-AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV-induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A-AS1 overexpression or HNF1A-AS1 knockdown. Taken together, these results revealed that HNF4A-AS1 and HNF1A-AS1 modulated RTV-induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter. SIGNIFICANCE STATEMENT: HNF4A-AS1 and HNF1A-AS1, transcribed separately from neighboring antisense genes of the human transcription factor genes HNF4A and HNF1A, were identified as long noncoding RNAs that can affect RTV-induced hepatotoxicity and susceptibility to RTV-induced hepatotoxicity caused by rifampicin exposure, mainly by affecting the expression of CY3A4 via alterations in PXR enrichment and histone modification status in the CYP3A4 promoter. This discovery provides directions for further research on the mechanisms of RTV-induced liver injury.

c-Fos is upregulated in the genital tubercle of DEHP-induced hypospadiac rats and the prepuce of patients with hypospadias.

This study aimed to investigate the expression of Fos proto-oncogene, AP-1 transcription factor subunit (c-Fos) in the genital tubercle (GT) of rats with di(2-ethylhexyl) phthalate (DEHP)-induced hypospadias and in the prepuce of patients with hypospadias compared with unaffected controls. Pregnant rats were given 750 mg/kg/day DEHP orally from gestational days 12-19. Western blotting showed that c-Fos expression was increased in DEHP-induced hypospadiac male offspring. In addition, 30 prepuce tissue specimens obtained during hypospadias repair surgery were divided into 2 groups: the mild hypospadias group (n = 15) and the severe hypospadias group (n = 15). Fifteen normal prepuce tissue specimens were harvested during elective circumcision as normal controls. Real-time quantitative polymerase chain reaction, western blotting and immunohistochemistry analyses were used to assess c-Fos expression. c-Fos protein levels were higher in the GT of DEHP-induced rats than in that of control rats. c-Fos mRNA and protein levels were also higher in the hypospadias groups than in the control group (p < 0.05, p < 0.001), and c-Fos protein levels were significantly higher in the severe hypospadias group than in the mild hypospadias group (p < 0.01). The expression of c-Fos was increased in both the GT of DEHP-induced hypospadiac rats and the prepuce of hypospadias patients. Thus, c-Fos overexpression might contribute to hypospadias.Abbreviations: DEHP: di(2-ethylhexyl) phthalate; c-Fos: Fos proto-oncogene, AP-1 transcription factor subunit; Mafb: the masculinization-regulatory gene v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B; GT: genital tubercle; ED: embryonic day; AGD: anogenital distance; AGI: anogenital distance index; ED: embryonic day.

Human umbilical cord mesenchymal stem cell attenuates renal fibrosis via TGF-ß/Smad signaling pathways in vivo and in vitro.

Renal fibrosis is a progressive pathological process that eventually leads to end-stage renal failure with limited therapeutic options. The aim of this study was to investigate the nephron-protective effect of human umbilical cord mesenchymal stem cells (ucMSCs) on renal fibrosis. UcMSCs were intravenously injected into renal fibrosis mice induced by aristolochic acid (AA) and co-cultured with HK-2 cells induced by TGF-ß1, respectively. The kidney functions including serum creatinine (Scr) and blood urea nitrogen (BUN) levels, and histopathology were examined after treated with stem cells and normal saline as control. Immunohistochemical staining, immunofluorescent staining, and Western blot analysis were used to assessed the expression of proteins associated with epithelial to mesenchymal transition (EMT) and TGF-ß/Smad signaling pathway. The results showed that ucMSCs effectively improved the kidney function and pathological structure, reduced AA-induced fibrosis and extracellular matrix deposition. Besides, UcMSCs significantly inhibited the EMT process and TGF-ß1/Smad signaling pathway in AA-induced mice and TGF-ß1-induced HK-2 cells compared to the control (p < 0.05). Our data suggested that ucMSCs play as a nephron-protective role in anti-fibrosis through inhibiting the activation of TGF-ß1/Smad signaling pathway.

Human ucMSCs seeded in a decellularized kidney scaffold attenuate renal fibrosis by reducing epithelial-mesenchymal transition via the TGF-ß/Smad signaling pathway.

BACKGROUND: Renal fibrosis occurs largely through epithelial-mesenchymal transition (EMT). This study explored the beneficial effects of a human umbilical cord mesenchymal stem cell-loaded decellularized kidney scaffold (ucMSC-DKS) on renal fibrosis in a rodent model of post-transplantation renal failure, and the underlying mechanism. METHODS: Rat-derived DKSs were examined after preparation, and then recellularized with human ucMSCs to prepare cell-loaded patches. A rat model of renal failure was established after subtotal nephrectomy (STN). The cell patches were transplanted to remnant kidneys. Changes in renal function, histology, EMT, and proteins related to the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in the remnant kidneys were examined 8 weeks after surgery, compared with non-cell patch controls. RESULTS: The DKSs were acellular and porous, with rich cytokine and major extracellular matrix components. The ucMSCs were distributed uniformly in the DKSs. Renal function was improved, renal fibrosis and EMT were reduced, and the TGF-ß/Smad signaling pathway was inhibited compared with controls at 8 weeks after ucMSC-DKS patch transplantation. CONCLUSIONS: The ucMSC-DKS restores renal function and reduces fibrosis by reducing EMT via the TGF-ß/Smad signaling pathway in rats that have undergone STN. It provides an alternative for renal fibrosis treatment.