Synbiotic regulates gut microbiota in patients with lupus nephritis: an analysis using metagenomic and metabolome sequencing.

Objective: To investigate the changes in gut microbes and their metabolites after administering synbiotics to patients with new-onset lupus nephritis (LN) treated using a conventional method and provide a theoretical basis for finding new targets for the diagnosis and treatment of LN. Methods: In this study, a total of 12 participants were divided into the lupus and synbiotic groups. Stool samples and clinical data were collected before and after treatment for metagenomic, nontargeted metabolomic, and statistical analyses. Results: The relative abundances of the pathogenic bacteria Prevotella, Bacteroides, and Enterobacteriaceae_unclassified decreased after synbiotic treatment, whereas the abundances of Actinobacteria and Firmicutes increased. Further, the Firmicutes to Bacteroidetes ratio increased; however, the difference was not statistically significant (p > 0.05). α diversity analysis showed no significant differences in the intestinal microbial richness and diversity index of patients with LN between the groups before and after treatment (p > 0.05). ß analysis showed the differences in the community structure between the samples of the two groups before and after treatment. Linear discriminant analysis effect size and receiver operating characteristic curve analyses revealed that Negativicutes (AUC = 0.9722) and Enterobacteriaceae_unclassified (AUC = 0.9722) were the best predictors of the lupus and synbiotic groups, respectively, before and after treatment. Joint analyses revealed that amino acid biosynthesis, aminoacyl-tRNA biosynthesis, purine metabolism, and other metabolic pathways may be involved in the changes in the metabolic function of patients with LN after the addition of synbiotics. Spearman's correlation analysis revealed the interaction between clinical features and flora, and flora exhibited a complex biological network regulatory relationship. Conclusion: Synbiotics regulate the metabolic functions of intestinal microorganisms in patients with LN and play a role in various biological functions. Synbiotic supplements may be safe and promising candidates for patients with LN.

FKN secreted by kidney epithelial cells regulates macrophage activation in lupus nephritis via the Hippo signaling pathway.

BACKGROUND: Lupus nephritis (LN) is a serious complication of systemic lupus erythematosus (SLE), and its pathogenesis is not fully understood. Previously, we showed that fractalkine (FKN) expression was positively correlated with the severity of LN. Here, we aimed to study the role of the Hippo signaling pathway (HSP) and its interaction with FKN in LN in an attempt to provide novel strategies for LN treatment. METHODS: In this study, lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-stimulated THP-1 cells were co-cultured with FKN up-regulated or down-regulated kidney epithelial cells Hkb20. FKN-knockout (KO-FKN) mice were used to construct LN model. Flow cytometric analysis, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), pathological staining, Western blot, and immunofluorescence (IF) staining were employed to investigate the role of FKN and its interaction with the Hippo signaling pathway (HSP) in LN. RESULTS: Up-regulation of FKN in kidney epithelial cells was associated with increased macrophage activation. FKN overexpression in kidney epithelial cells suppressed apoptosis, inflammation levels, and M1 polarization of THP-1 cells and inhibited the HSP. Oppositely, FKN knockdown in kidney epithelial cells increased apoptosis, inflammation, and M1 polarization and activated the HSP. HSP inhibitor reversed the effect of FKN knockdown on THP-1 cells. In LN mice, FKN knockout and YAP inhibitor decreased the levels of renal function markers, alleviated kidney injury induced by LN, and inhibited macrophage activation in LN mice. CONCLUSIONS: FKN down-regulation reduced the activation of macrophages in renal tissue and alleviated kidney damage by activating HSP. The regulatory effect of FKN on HSP should be confirmed in patients with LN, and the mechanism of FKN in LN should be further explored.

Calcineurin inhibitors or cyclophosphamide in the treatment of membranous nephropathy superimposed with FSGS lesions: a retrospective study from China.

BACKGROUND: Cyclophosphamide (CTX) and calcineurin inhibitors (CNIs) based regimens are recommended as immunosuppressive therapies for patients with idiopathic membranous nephropathy (IMN). Focal and segmental glomerular sclerosis (FSGS) lesions, which are common in membranous nephropathy (MN), are poor predictors of outcome. This study compared the differences of prognosis between two regimens in patients with IMN combined with FSGS lesions. METHODS: This retrospective study enrolled 108 patients with biopsy-proven IMN, accompanied with FSGS lesions, nephrotic syndrome and an estimated glomerular filtration rate (eGFR)≥60 mL/min/1.73 m2 who were treated with CTX or CNIs. We used propensity score matching (PSM) for balancing the confounding variables. RESULTS: During follow-up, 10 patients (10/55 [18.2%]; nine males) in the CNIs group showed a 50% decline in eGFR; eight had a not otherwise specified variant. Patients initially treated with CNIs had a significantly higher risk of progression to the primary outcome and a lower probability of complete or total remission. The relapse rate was higher in patients who initially received CNIs- than in those who received CTX-based treatment. Before PSM, age and 24-h urine protein level differed significantly between the groups. The PSM model included data from 72 patients. Worse outcomes were also noted among patients who initially received CNIs than those who received CTX-based treatments after matching. CONCLUSIONS: Patients with MN combined with FSGS lesions have a higher risk of renal functional decline and a higher rate of relapse after CNIs than after CTX therapy.

HOXA11-OS participates in lupus nephritis by targeting miR-124-3p mediating Cyr61 to regulate podocyte autophagy.

BACKGROUND: The long chain non-coding RNA HOXA11-OS was recently identified. Increasing studies have shown that HOXA11-OS has regulatory effects on genes in gastric cancer, prostate cancer, and various kidney diseases, but research on its role in systemic lupus erythematosus is still lacking. The present study aimed to investigate the role of HOXA11-OS in the regulation of podocyte autophagy in the development of lupus nephritis (LN) and its potential molecular mechanism. METHODS: mRNA and protein expression of the target gene (i.e., Cyr61) was detected by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. Mouse podocytes were induced using serum immunoglobulin G (IgG) from patients with lupus and their viability was detected using the cell counting kit-8 assay. The interaction of miR-124-3p with HOXA11-OS and Cyr61 was analyzed by double luciferase reporter gene assay. Serum autoantibody levels were detected by enzyme-linked immunosorbent assay. Pathological lesions in the kidney tissue were detected by hematoxylin-eosin and periodate-Schiff staining. The independent samples t-test was used for comparing two groups, and one-way analysis of variance for comparing multiple groups. RESULTS: HOXA11-OS was highly expressed in LN tissues, serum, and cells, and the expression of some key autophagy factors and Cyr61 was significantly increased, while miR-124-3p expression was significantly decreased. In vitro, LN-IgG inhibited podocyte activity, increased autophagy and Cyr61 expression, and aggravated podocyte injury in a time- and dose-dependent manner. As a competitive endogenous RNA of miR-124-3p, HOXA11-OS promoted the expression of Cyr61, thus enhancing the autophagy increase induced by LN-IgG and aggravating podocyte injury. Knockdown of HOXA11-OS had the opposite effect. miR-124-3p mimic or Cyr61 knockdown restored the high expression of autophagy factors and Cyr61 induced by HOXA11-OS overexpression and alleviated podocyte injury. Further in vivo experiments showed that injection of sh-HOXA11-OS adeno-associated virus downregulated HOXA11-OS and significantly alleviated renal damage in lupus mice. CONCLUSIONS: HOXA11-OS is involved in the occurrence and development of LN by regulating podocyte autophagy through miR-124-3p/Cyr61 sponging, which may provide a good potential therapeutic target for LN.

Fractalkine deficiency attenuates LPS-induced acute kidney injury and podocyte apoptosis by targeting the PI3K/Akt signal pathway.

BACKGROUND: Podocyte injury is a major biomarker of primary glomerular disease, which leads to massive proteinuria and kidney failure. The increased production of the chemokine, fractalkine (FKN, CX3CL1), is a hallmark of multiple inflammatory diseases. However, the underlying mechanism of FKN in podocyte injury remains unknown. METHODS: In this study, we performed an LPS infusion model in FKN knockout (FKN-/-, FKN-KO) mice. In cultured podocytes, we used plasmids to knockdown FKN and treated the podocytes with PI3K/Akt inhibitor (LY294002). Haematoxylin and eosin (HE) staining, Western Bolt, Co-immunoprecipitation (Co-IP), Immunofluorescence staining and flow cytometric analysis were employed to establish the role of FKN in podocyte injury. RESULTS: LPS stimulation resulted in kidney damage, increased the expression of the Bcl-2 family apoptosis protein, and decreased podocyte marker protein (nephrin, podocin and WT1) abundance compared with the WT mice. LPS-induced FKN-KO mice exhibited reduced lethality and inflammatory cell infiltration, podocyte apoptosis, and PI3K/Akt signal pathway inhibition compared to WT mice. In cultured podocytes, the interaction between FKN and the PI3K/Akt signalling pathway was well confirmed. FKN knockdown reduced podocyte apoptosis by regulating the Bcl-2 family; however, this protective effect was reversed by the co-administration of a PI3K/Akt inhibitor (LY294002). CONCLUSION: Overall, these findings reveal a novel mechanistic property of FKN, PI3K/Akt signalling, and podocyte apoptosis.

[CX3CL1/fractalkine inhibits lipopolysaccharide-induced apoptosis of mouse RAW264.7 macrophages by activating Wnt/ß-catenin signal pathway].

Objective To investigate the mechanism of CX3CL1/fractalkine (FKN) in lipopolysaccharide (LPS)-induced apoptosis of mouse RAW264.7 macrophages. Methods RAW264.7 macrophages were infected with FKN overexpression or knockdown lentivirus plasmids containing red fluorescent protein and treated with LPS. The apoptosis of RAW264.7 macrophages was detected by flow cytometry. The expression levels of FKN, Wnt4, ß-catenin, cleaved caspase-3(c-caspase-3), c-caspase-9, BAX and cytochrome C (CytC) proteins were measured by Western blotting. The expression and localization of c-caspase-3 and c-caspase-9 in RAW264.7 macrophages were determined by immunofluorescence cytochemistry. Results Compared with control group, the apoptosis rate and the protein levels of FKN, Wnt4, ß-catenin, c-caspase-3, c-caspase-9, BAX and CytC increased significantly in LPS group. Compared with LPS group, the apoptosis rate of FKN overexpression combined with LPS group was significantly decreased. The protein levels of FKN, Wnt4 and ß-Catenin reported an increase, while the protein levels of c-caspase-3, c-caspase-9, BAX, CytC and localization of c-caspase-3 and c-caspase-9 in the cytoplasm showed a decrease in FKN overexpression combined with LPS group. The opposite results were observed in FKN knockdown combined with LPS group. Conclusion CX3CL1/FKN can activate Wnt/ß-catenin signal pathway, downregulate the key proteins expression of mitochondrial apoptosis pathway, and reduce LPS-induced apoptosis of RAW264.7 macrophages.

CYR61, regulated by miR-22-3p and MALAT1, promotes autophagy in HK-2 cell inflammatory model.

BACKGROUND: Renal tubular epithelial cells play an important role in renal function and are a major site of injury from inflammation. Emerging evidence suggests that CYR61 is involved in the regulation of autophagy. However, there are few studies on CYR61 in nephropathy and associated inflammation. This study aimed to clarify how CYR61 regulates autophagy in human renal epithelial cells while in an inflammatory state and regulates the upstream pathway of CYR61 levels. METHODS: The human renal tubular epithelial cells (HK-2) cell line treated by lipopolysaccharide (LPS) was used as an inflammatory model of human epithelial cells. Short hairpin RNA (shRNA) was used to down-regulate CYR61, and the changes in the transcription and expression levels of related molecules, as well as the morphological changes of HK-2 cells, were detected by quantitative real time-PCR (qRT-PCR), western blot (WB), and transmission electron microscopy. Either CYR61 or MALAT1 were up-regulated by overexpression vectors, or MALAT1 was down-regulated by miR-22-3p mimics. Subsequently, the levels of CYR61, MALAT1, related inflammatory factors, and autophagy factors were measured by qPCR, WB, and enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry and acridine-orange assay. RESULTS: We observed that down-regulation of CYR61 could down-regulate 1B-light chain 3 (LC3) level and inhibit autophagy in the LPS-induced inflammation model of HK-2 cells. The expression levels of CYR61, Beclin1, Atg5, LC3, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were significantly increased by upregulating CYR61 or MALAT1 by overexpression vector, while the expression level of p62 was significantly decreased, intracellular reactive oxygen species (ROS) content was increased, and the proportion of autophagy and apoptosis was increased. The use of miR-22-3p mimics significantly reversed the changes induced by up-regulation of CYR61 or MALAT1 at the molecular and cellular levels. CONCLUSIONS: Our data indicated that CYR61 positively regulates autophagy of HK-2 cells under an inflammatory state, and was negatively regulated by miR-22-3p, while miR-22-3p and MALAT1 were negatively regulated by each other.

Circular RNA-0007059 protects cell viability and reduces inflammation in a nephritis cell model by inhibiting microRNA-1278/SHP-1/STAT3 signaling.

BACKGROUND: Increasing evidence has indicated that circular RNAs (circRNAs) play a role in various diseases. However, the influence of circRNAs in nephritis remains unknown. METHODS: Microarray analysis and RT-qPCR were used to detect the expression of circRNA. Type I IFN were administrated to RMC and HEK293 cells to establish a nephritis cell model. CCK-8, MTT assay, and flow cytometry were used to assess cell proliferation, viability, and apoptosis of cells. Bioinformatics analysis and dual luciferase reporter assay detect the interaction of circ_0007059, miRNA-1278, and SHP-1. Glomerulonephritis was performed in a mouse model by administration of IFNα-expressing adenovirus. IHC staining showed the pathogenic changes. RESULTS: In the present study, the expression of circ_0007059 in type I interferon (IFN)-treated renal mesangial cells (RMCs), lupus nephritis (LN) specimens, and HEK293 cells was downregulated compared with that in normal healthy samples and untreated cells. Circ_0007059 overexpression resulted in increased cell proliferation, cell viability, apoptosis, and inflammation-associated factors (CXCL10, IFIT1, ISG15, and MX1) in RMCs and HEK293 cells. In addition, circ_0007059 overexpression significantly restored cell proliferation and viability and inhibited IFN-induced apoptosis. Further, the increased expression resulted in reduced inflammation and the downregulation of CXCL10, IFIT1, ISG15, and MX1 in RMCs and HEK293 cells. Circ_0007059 serves as a sponge for miR-1278 so that the latter can target the 3'-untranslated region of SHP-1. Overexpressed circ_0007059 inhibited miR-1278 expression and elevated SHP-1 expression, subsequently reducing STAT3 phosphorylation. Meanwhile, miR-1278 was upregulated and SHP-1 was downregulated in LN samples and IFN-treated cells. The restoration of miR-1278 counteracted the effect of circ_0007059 on viability, apoptosis, and inflammation as well as on SHP-1/STAT3 signaling in RMCs and HEK293 cells. We also investigated the role of SHP-1 overexpression in IFN-treated RMCs and HEK293 cells; SHP-1 overexpression resulted in a similar phenotype as that observed with circ_0007059 expression. CONCLUSIONS: The study indicates that circ_0007059 protects RMCs against apoptosis and inflammation during nephritis by attenuating miR-1278/SHP-1/STAT3 signaling.

Depletion of Fractalkine ameliorates renal injury and Treg cell apoptosis via the p38MAPK pathway in lupus-prone mice.

Fractalkine (FKN) is a chemokine with several roles, including chemotaxis; adhesion; and immune damage, which also participates in cell inflammation and apoptosis and responds to the pathogenesis of autoimmune diseases. Given the involvement of regulatory T cells (Treg) cells in autoimmune diseases, this study investigated the regulatory mechanism of FKN in renal injury and Treg apoptosis via the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in lupus-prone mice. Lupus was induced in BALB/c female mice by injection of pristane, followed by isolation of CD4+CD25+ Treg cells from the spleen of lupus model mice. To deplete FKN, mice received injection of an anti-FKN antibody, and Treg cells were transfected with FKN small-interfering RNA. Lupus mice and Treg cells were treated with the p38MAPK inhibitor SB203580 and activator U-46619, respectively, and urine protein and serum urea nitrogen, creatinine, and autoantibodies were measured and renal histopathological changes analyzed. We determined levels of FKN, phosphorylated p38 (p-p38), and forkhead box P3 (FOXP3) in renal tissue and Treg cells, and analyzed apoptosis rates and levels of key apoptotic factors in Treg cells. The renal FKN and p-p38 levels increased, whereas renal FOXP3 level decreased in lupus-prone mice. Treatment with the anti-FKN antibody and the p38MAPK inhibitor ameliorated proteinuria and renal function, significantly reducing serum autoantibody, renal FKN, and p-p38 levels while increasing renal FOXP3 level in lupus-prone mice. Moreover, FKN knockdown and administration of the p38MAPK inhibitor reduced apoptosis and levels of pro-apoptotic factors, increased levels of anti-apoptotic factors, and suppressed activation of p38MAPK signaling in Treg cells derived from lupus model mice. Furthermore, treatment with the p38MAPK activator U-46619 had the opposite effect on these cells. These data indicated that depletion of FKN ameliorated renal injury and Treg cell apoptosis via inhibition of p38MAPK signaling in lupus nephritis, suggesting that targeting FKN represents a potential therapeutic strategy for treating Lupus nephritis.

Fractalkine aggravates LPS-induced macrophage activation and acute kidney injury via Wnt/ß-catenin signalling pathway.

Fractalkine (CX3CL1, FKN), a CX3C gene sequence inflammatory chemokine, has been found to have pro-inflammatory and pro-adhesion effects. Macrophages are immune cells with a critical role in regulating the inflammatory response. The imbalance of M1/M2 macrophage polarization can lead to aggravated inflammation. This study attempts to investigate the mechanisms through which FKN regulates macrophage activation and the acute kidney injury (AKI) involved in inflammatory response induced by lipopolysaccharide (LPS) by using FKN knockout (FKN-KO) mice and cultured macrophages. It was found that FKN and Wnt/ß-catenin signalling have a positive interaction in macrophages. FKN overexpression inhibited LPS-induced macrophage apoptosis. However, it enhanced their cell viability and transformed them into the M2 type. The effects of FKN overexpression were accelerated by activation of Wnt/ß-catenin signalling. In the in vivo experiments, FKN deficiency suppressed macrophage activation and reduced AKI induced by LPS. Inhibition of Wnt/ß-catenin signalling and FKN deficiency further mitigated the pathologic process of AKI. In summary, we provide a novel mechanism underlying activation of macrophages in LPS-induced AKI. Although LPS-induced murine AKI was unable to completely recapitulate human AKI, the positive interactions between FKN and Wnt/ß-catenin signalling pathway may be a therapeutic target in the treatment of kidney injury.

[Fractalkine inhibits lipopolysaccharide-induced M1 polarization of macrophages by activating Wnt/ß-catenin signaling pathway].

OBJECTIVE: To explore the mechanism by which fractalkine (CX3CL1; FKN) inhibits lipopolysaccharide (LPS)-induced immunological response in RAW264.7 cells. METHODS: A RAW264.7 cell model overexpressing FKN was established by transfection with the lentiviral vector CX3CL1. The effects of LPS, ICG-001 (a Wnt/ß-catenin signaling pathway inhibitor), either alone or in combination, on M1 polarization of na?ve and FKN-overexpressing RAW264.7 cells were evaluated by detecting of intereukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) using ELISA. The protein expressions of the inflammatory factors (iNOS, TNF-α, and IL-6), FKN, Wnt-4, and ß-catenin were detected by Western blotting. The subcellular localization of IL-6 in the cells was detected by immunofluorescence assay. RESULTS: The RAW264.7 cell model of FKN overexpression was successfully established. In na?ve RAW264.7 cells, treatment with both ICG-001 and LPS, as compared with LPS alone, significant promoted TNF-α and IL-6 secretions, increased intracellular levels of TNF-α, IL-6 and iNOS (P < 0.05), and reduced intracellular FKN, Wnt-4 and ß-catenin levels (P < 0.01). In FKN-overexpressing RAW264.7 cells, LPS treatment significantly reduced the secretion of TNF-α and IL-6 and intracellular levels of TNF-α, IL-6 and iNOS (P < 0.01), increased intracellular FKN, Wnt-4 and ß-catenin protein contents (P < 0.01), and inhibited IL-6 localization in the cytoplasm; compared with LPS, the combined treatment with ICG-001 and LPS obviously enhanced IL-6 localization in the cytoplasm of the cells. CONCLUSIONS: FKN overexpression suppresses LPS-induced M1 type polarization of RAW264.7 cells by activating Wnt/ß-catenin signaling pathway.

Fractalkine mediates lymphocyte inflammation and tubulointerstitial lesions by modifying the Treg/Th17 balance in lupus-prone MRL/lpr mice.

In this study, we first analyzed the expression level of fractalkine (FKN) in the serum of patients with lupus nephritis (LN) and the distribution of peripheral blood Treg cells, and explored FKN and Treg cells, systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) correlation. Subsequently, we explored the specific role of FKN in tubule interstitial lesions (TILs) and regulatory T (Treg) cells/T helper (Th) 17 cell balance in lupus model mice. Treated with an anti-FKN antibody (aFKN), recombinant FKN (rFKN), or an isotype antibody (IgG) in MRL/MpJ-Faslpr/J and C57BL/6 mice, and then detected TIL level and forkhead box p3 (Foxp3), IL-10, IL-17 and IL-6 expression levels in the kidney and spleen in the proportion of Treg and Th17 cells. Finally, then use aFKN, rFKN, or IgG to intervene in polarized Tregs with IL-6, TGF-ß, IL-23, anti-interferon, and Th17 cells with anti-IL-4 after transforming to transform growth factor (TGF)-ß and interleukin (IL)-2 in isolated mouse spleen lymphocytes. The results showed that the expression level of FKN was positively correlated with SLEDAI-2K and negatively correlated with the distribution of Treg cells. After treatment with aFKN in lupus model mice, kidney damage was delayed, TIL formation was reduced, Foxp3 and IL-10 levels were up-regulated, IL-17 and IL-6 levels were down-regulated in renal tissues, Th17 cell subsets and Treg cell subsets were reduced The increase is in the spleen, and rFKN treatment has the opposite effect in mouse. In addition, after interfering with polarized cells by aFKN, it was found that IL-17 and IL-6 expression levels were down-regulated in Th17 cells, Foxp3 and IL-10 levels in Tregs were up-regulated, and rFKN treatment had the opposite effect in vitro. These results indicate that FKN participates in and promotes SLE target organ damage including: secretion of inflammatory factors and renal TIL, and most importantly, these effects might have been due to modification of the Treg/Th17 cell balance.

Critical roles of PI3K/Akt/NF­κB survival axis in angiotensin II­induced podocyte injury.

Numerous studies have reported that angiotensin (Ang) II, nephrin, and podocin serve pivotal roles in podocyte injury, and thus can lead to the occurrence of proteinuria and the progression of kidney diseases. This study aimed to investigate the effects of Ang II on the production of nephrin and podocin, and their relationship with podocyte injury. We also aimed to determine whether nephrin, podocin and caspase­9 production depends on the PI3K/Akt/nuclear factor (NF)­κB signaling pathway in cultured mouse podocytes. We treated mouse podocytes with different doses of Ang II (10­9, 10­8, 10­7 and 10­6 mol/l) for 12, 24, and 48 h to analyse cell viability, and at 10­6 mol/l Ang II for 12, 24, and 48 h to evaluate cell apoptosis. Cells were treated with 10­6 mol/l of Ang II and/or LY294002 (inhibitor of Akt) or 740Y­P (activator of PI3K) for 48 h to detect Akt, phosphorylated (phospho)­Akt, p65 NF­κB, and phospho­p65 NF­κB, nephrin, podocin and caspase­9 expression, and podocyte apoptosis. Treatment with Ang II suppressed the viability and promoted the apoptosis of podocytes in a dose­ and time­dependent manner. Ang II decreased phospho­Akt, phospho­p65 NF­κB, nephrin, and podocin and increased caspase­9 expression, while podocyte apoptosis was promoted. LY294002 further enhanced Ang II­induced downregulation of Akt and p65 NF­κB activation, as well as upregulation of caspase­9 mRNA and protein, and promoted the apoptosis of podocytes. Of note, 740Y­P restored Ang II­induced downregulation of Akt and p65 NF­κB activation, and upregulation of caspase­9, and decreased podocyte apoptosis. Interestingly, LY294002 and 740Y­P were determined to have no notable effects on the expression of nephrin and podocin. The data suggested that Ang II could regulate the expression of nephrin, podocin and caspase­9. Collectively, our findings suggested that the PI3K/Akt/NF­κB survival axis may serve a pivotal role in podocyte injury.

Vasoactive intestinal peptide ameliorates renal injury in a pristane-induced lupus mouse model by modulating Th17/Treg balance.

BACKGROUND: Lupus nephritis (LN) is an inflammation of the kidneys and is a major cause of mortality in systemic lupus erythaematosus (SLE) patients. In addition, Th17/Treg balance is one of the most important factors that can promote the development of LN. It has been reported that vasoactive intestinal peptide (VIP) is associated with the downregulation of both inflammatory and autoimmune diseases through regulating T lymphocyte balance. Therefore, the aim of this study was to determine the role of VIP in modulating Th17/Treg balance in LN. METHODS: LN was induced in BALB/c female mice by injection pristane. After 3 months, mice were randomly divided into four groups: control, VIP + control, LN and VIP + LN. Autoantibody levels were tested by ELISA. The distribution of Th17/Treg cells in vivo and in vitro was detected by FC. Renal tissues were examined by PASM and DIF for pathology and Foxp3+CD3+. The mRNA and protein expression levels of pro- and anti-inflammatory cytokines were detected by qRT-PCR and western blotting. RESULTS: VIP can improve renal injury by regulating Th17/Treg imbalance in LN mice. Proteinuria, renal function defects and autoantibodies were significantly decreased, and Th17/Treg cell balance was restored in VIP compared with LN mice. In addition, VIP improved renal lesions by promoting the expression of Foxp3+CD3+ in renal tissue. Furthermore, VIP downregulated the mRNA and protein expression of IL-17, IL-6 and upregulated Foxp3, IL-10 expression. CONCLUSIONS: VIP reduced LN proteinuria and renal function defects and restored the Th17/Treg cell balance. Furthermore, VIP also downregulated autoantibody and inflammatory cytokine expression and upregulated Foxp3 and IL-10 expression.

The Predictive Value of Autoantibody Spectrum on Organ Damage in Patients With Systemic Lupus Erythematosus.

OBJECTIVES: This study aims to investigate the positive detection rate and predictive value of autoantibodies, including anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibodies, anti-histone antibodies (AHAs), anti-ribosomal (anti-Rib) P antibodies, anti-Smith (anti-Sm) antibodies, anti-U1 ribonucleoprotein (anti-U1RNP) antibodies, anti-Sjögren's syndrome type A antibodies and anti-Sjögren's syndrome type B antibodies, on organ damage in patients with systemic lupus erythematous (SLE). PATIENTS AND METHODS: A total of 225 patients with SLE (37 males, 188 females; mean age 37.4±15.9 years; range, 7 to 80 years) were evaluated retrospectively. Statistical analysis was performed to obtain the positive detection rate of autoantibodies and to investigate the predictive value. RESULTS: There were statistically significant differences of positive anti-dsDNA antibodies in renal damage, photosensitization, hematological abnormalities and serositis (p<0.05) and a statistically significant difference of positive AHAs in photosensitization (p<0.05). There was statistically significant difference of positive anti-U1RNP antibodies in renal damage (p<0.05). There were also statistically significant differences of positive anti-Smith antibodies in renal damage, arthritis, photosensitization, oral ulcers, hematological abnormalities and serositis (p<0.05) and of positive anti-Rib antibodies in renal damage, arthritis, photosensitization, malar rash, hematological abnormalities and serositis (p<0.05). However, there were no statistically significant differences of positive anti-Sjögren's syndrome type B antibodies and anti-Sjögren's syndrome type A antibodies in renal damage, arthritis, malar rash, neuropsychiatric disorders, hematological abnormalities and serositis (p>0.05). CONCLUSION: Autoantibody spectrum is an important serological basis for SLE diagnosis. There are differences in the autoantibodies distribution of SLE patients with different organ damage, suggesting a certain clinical value for prediction of organ damage in SLE.

[Angiotensin â ¡ induces apoptosis of HEK293T cells by up-regulating Cyr61 expression].

OBJECTIVE: To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism. METHODS: Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10-7 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting. RESULTS: The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells (P < 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group (P < 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% vs (3.87 ± 0.83)%, P < 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment (P < 0.05). CONCLUSIONS: The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.

Identification of the Transcriptional Networks and the Involvement in Angiotensin II-Induced Injury after CRISPR/Cas9-Mediated Knockdown of Cyr61 in HEK293T Cells.

BACKGROUND: The transcriptional networks of Cyr61 and its function in cell injury are poorly understood. The present study depicted the lncRNA and mRNA profiles and the involvement in angiotensin II-induced injury after Cyr61 knockdown mediated by CRISPR/Cas9 in HEK293T cells. METHODS: HEK293T cells were cultured, and Cyr61 knockdown was achieved by transfection of the CRISPR/Cas9 KO plasmid. lncRNA and mRNA microarrays were used to identify differentially expressed genes (DEGs). Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine biofunctions and signaling pathways. RT-PCR was used to validate the microarray results. Cells were divided into four groups: control, Cyr61 knockdown, angiotensin II (Ang II) without Cyr61 knockdown, and Ang II with Cyr61 knockdown. CCK8, western blotting, and flow cytometry analysis were carried out to dissect cellular function. RESULTS: A total of 23184 lncRNAs and 28264 mRNAs were normalized. 26 lncRNAs and 212 mRNAs were upregulated, and 74 lncRNAs and 233 mRNAs were downregulated after Cyr61 knockdown. Analysis of cellular components, molecular functions, biological processes, and regulatory pathways associated with the differentially expressed mRNAs revealed downstream mechanisms of the Cyr61 gene. The differentially expressed genes were affected for small cell lung cancer, axon guidance, Fc gamma R-mediated phagocytosis, MAPK signaling pathway, focal adhesion, insulin resistance, and metabolic pathways. In addition, Cyr61 expression was increased in accordance with induction of cell cycle arrest and apoptosis and inhibition of cell proliferation induced by Ang II. Knockdown of Cyr61 in HEK293T cells promoted cell cycle procession, decreased apoptosis, and promoted cell proliferation. CONCLUSIONS: The Cyr61 gene is involved in Ang II-induced injury in HEK293T cells. Functional mechanisms of the differentially expressed lncRNAs and mRNAs as well as identification of metabolic pathways will provide new therapeutic targets for Cyr61-realated diseases.

FKN Facilitates HK-2 Cell EMT and Tubulointerstitial Lesions via the Wnt/ß-Catenin Pathway in a Murine Model of Lupus Nephritis.

Fractalkine (FKN), also known as chemokine (C-X3-C motif) ligand 1, constitutes an intriguing chemokine with a documented role in the development of numerous inflammatory diseases including autoimmune disease. Specifically, it has been reported that FKN is involved in the disease progression of lupus nephritis (LN). The epithelial-mesenchymal transition (EMT) plays a significant role in the formation of tubulointerstitial lesions (TIL), which are increasingly recognized as a hallmark of tissue fibrogenesis after injury. However, the correlation between FKN and EMT or TIL in LN has not been determined. To investigate the potential role of FKN in EMT and TIL, MRL lymphoproliferation (MRL/lpr) strain mice were treated with an anti-FKN antibody, recombinant-FKN chemokine domain, or isotype antibody. Our results revealed that treatment with the anti-FKN antibody improved EMT, TIL, and renal function in MRL/lpr mice, along with inhibiting activation of the Wnt/ß-catenin signaling pathway. In contrast, administration of the recombinant-FKN chemokine domain had the opposite effect. Furthermore, to further explore the roles of FKN in EMT, we assessed the levels of EMT markers in FKN-depleted or overexpressing human proximal tubule epithelial HK-2 cells. Our results provide the first evidence that the E-cadherin level was upregulated, whereas α-SMA and vimentin expression was downregulated in FKN-depleted HK-2 cells. In contrast, overexpression of FKN in HK-2 cells enhanced EMT. In addition, inhibition of the Wnt/ß-catenin pathway by XAV939 negated the effect of FKN overexpression, whereas activation of the Wnt/ß-catenin pathway by Ang II impaired the effect of the FKN knockout on EMT in HK-2 cells. Together, our data indicate that FKN plays essential roles in the EMT progression and development of TIL in MRL/lpr mice, most likely through activation of the Wnt/ß-catenin signaling pathway.

Fractalkine is Involved in Lipopolysaccharide-Induced Podocyte Injury through the Wnt/ß-Catenin Pathway in an Acute Kidney Injury Mouse Model.

Injury to podocytes leads to proteinuria, a hallmark of most glomerular diseases as well as being associated with the progression of kidney disease. Activation of the Wnt/ß-catenin pathway is associated with the pathogenesis of podocyte dysfunction and can play a role in renal injury. Furthermore, the expression of fractalkine (FKN) induced by lipopolysaccharides (LPS) is also one of crucial inflammation factors closely related to renal tissue damage. The aim of this study is to explore the mechanism of LPS-induced FKN expression leading to podocyte injury and contribute to acute kidney injury (AKI) through regulation of Wnt/ß-catenin pathway. An AKI model was established for in vivo experiments and blood was collected for serum BUN and Cr measurement, and histopathological features of the kidneys were studied by PASM and IHC staining. For in vitro experiments, a mouse podocyte cell line was stimulated with different concentrations of LPS for 24 and 48 h after which podocyte viability and apoptosis of cells were evaluated. The expression of podocyte-specific markers, FKN and Wnt/ß-catenin pathway mRNA and protein was detected in mice and cells by using qRT-PCR and western blotting. LPS induced the expression of FKN and activation of the Wnt/ß-catenin pathway, leading to a decrease of podocyte-specific proteins which resulted in poor renal pathology and dysfunction in the AKI mouse model. Moreover, LPS treatment significantly decreased cell viability and induced podocyte apoptosis in a dose-dependent manner that causes changes in the expression of podocyte-specific proteins through activation of FKN and the Wnt/ß-catenin pathway. Thus, the expression of FKN and Wnt/ß-catenin pathway by LPS is closely associated with podocyte damage or loss and could therefore account for progressive AKI. Our findings indicate that LPS induce podocyte injury and contribute to the pathogenesis of AKI by upregulating the expression of FKN and Wnt/ß-catenin pathway.