Activation of BZW1 by CEBPB in macrophages promotes eIF2α phosphorylation-mediated metabolic reprogramming and endoplasmic reticulum stress in MRL/lpr lupus-prone mice.

BACKGROUND: Lupus nephritis (LN) is associated with significant mortality and morbidity, while effective therapeutics and biomarkers are limited since the pathogenesis is complex. This study investigated the roles of the CEBPB/BZW1/eIF2α axis in metabolic reprogramming and endoplasmic reticulum stress in LN. METHOD: The differentially expressed genes in LN were screened using bioinformatics tools. The expression of CEBPB in the renal tissue of patients with LN and its correlation with the levels of creatinine and urinary protein were analyzed. We used adenoviral vectors to construct LN mice with knockdown CEBPB using MRL/lpr lupus-prone mice and analyzed the physiological and autoimmune indices in mice. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and dual-luciferase reporter assays were conducted to explore the regulation of BZW1 by CEBPB, followed by glycolytic flux analysis, glucose uptake, and enzyme-linked immunosorbent assay (ELISA). Finally, the role of eIF2α phosphorylation by BZW1 in bone marrow-derived macrophages (BMDM) was explored using eIF2α phosphorylation and endoplasmic reticulum stress inhibitors. RESULTS: CEBPB was significantly increased in renal tissues of patients with LN and positively correlated with creatinine and urine protein levels in patients. Downregulation of CEBPB alleviated the autoimmune response and the development of nephritis in LN mice. Transcriptional activation of BZW1 by CEBPB-mediated glucose metabolic reprogramming in macrophages, and upregulation of BZW1 reversed the mitigating effect of CEBPB knockdown on LN. Regulation of eIF2α phosphorylation levels by BZW1 promoted endoplasmic reticulum stress-amplified inflammatory responses in BMDM. CONCLUSION: Transcriptional activation of BZW1 by CEBPB promoted phosphorylation of eIF2α to promote macrophage glycolysis and endoplasmic reticulum stress in the development of LN.

Knockdown of DEC2 expression inhibits the proliferation of mesangial cells through suppressed glycolysis and p38 MAPK/Erk pathway in lupus nephritis.

BACKGROUND: To elucidate the mechanism by which DEC2 modulates the proliferation of mesangial cells (MCs) in lupus nephritis (LN). METHODS: The 32-week-old female Fcgr2b-/- mice and their serum-treated MCs were used as in vivo and in vitro LN model. MCs knocked down of DEC2 and overexpressed with DEC2 were also established. The expression of DEC2 was measured in the kidneys of Fcgr2b-/- mice and LN serum-treated MCs using RT-qPCR and Western blot. MCs proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) labeling assay and PCNA expression using immunofluorescence. The glucose metabolism was evaluated in LN serum-treated MCs, and the levels of lactate production, glucose consumption, ATP production and mitochondrial membrane potential were assayed. The glycolysis and mitochondrial respiration function of the MCs were measured using the Extracellular Flux Analyzer. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were dynamically monitored and multiple important bioenergetic parameters can be calculated. The expression of Toll like receptor 4 (TLR4) and glucose transporter 1 (GLUT1) were detected in the MCs. Multiple signaling proteins were screened. RESULTS: DEC2 was found overexpressed in the kidney of Fcgr2b-/- LN mice. Knockdown of DEC2 inhibited LN serum-induced MCs proliferation. DEC2 was associated with the glucose metabolism in LN serum-treated MCs. DEC2 regulated glycolysis in LN serum-treated MCs. DEC2 was associated with mitochondrial fitness in LN serum treated MCs. DEC2 activated MCs glycolysis through TLR4 and glucose transporter 1 (GLUT1) regulation. DEC2 regulated MCs proliferation through two signaling pathways including dependent and independent of glycolysis, which located in the downstream of TLR4 signaling. CONCLUSION: Knockdown of DEC2 expression inhibits the proliferation of MCs through suppressed glycolysis and p38 MAPK/ERK pathway in LN.

A long non-coding RNA H19/microRNA-138/TLR3 network is involved in high phosphorus-mediated vascular calcification and chronic kidney disease.

Vascular calcification, characterized by the accumulation of calcium-phosphate crystals in blood vessels, is a major cause of cardiovascular complications and chronic kidney disease (CKD)-related death. This work focuses on the molecules involved in high-phosphorus-mediated vascular calcification in CKD. A rat model of CKD was established by 5/6 nephrectomy, and the rats were given normal phosphorus diet (NPD) or high phosphorus diet (HPD). HPD decreased kidney function, increased the concentration of calcium ion and damaged vascular structure in the thoracic aorta of diseased rats. A high phosphorus condition enhanced calcium deposition in vascular smooth muscle cells (VSMCs). High phosphorus also increased the expression of RUNX2 whereas reduced the expression of α-SM actin in the aortic tissues and VSMCs. Long non-coding RNA (lncRNA) H19 was upregulated in the aortic tissues after HPD treatment. H19 bound to microRNA (miR)-138 to block its inhibitory effect on TLR3 mRNA and activated the NF-κB signaling pathway. Downregulation of H19 or TLR3 alleviated, whereas downregulation of miR-138 aggravated the calcification and vascular damage in model rats and VSMCs. In conclusion, this study demonstrates that the H19/miR-138/TLR3 axis is involved in high phosphorus-mediated vascular calcification in rats with CKD.

TLR4 regulates vascular smooth muscle cell proliferation in hypertension via modulation of the NLRP3 inflammasome.

Backgroud: Toll-like receptor 4 (TLR4), a key mediator of inflammatory responses, which is associated with vascular remodeling. The association between TLR4 and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the regulation of vascular smooth muscle cell (VSMC) proliferation remains unclear. This study was to explore the role and underlying mechanisms of TLR4 in the proliferation of VSMC in hypertension. METHODS: VSMC proliferation after TLR4 overexpression or downregulation was determined by CCK-8, EdU Incorporation and colony formation assays. Western blots were carried out to investigate the expression of TLR4 and NLRP3 inflammasome components in VSMCs. Next, blood pressure measurements and Hematoxylin and Eosin (HE) staining assays were performed in spontaneously hypertensive rats (SHR). Media thickness (M) and diameter lumen (L) were measured as indicators of vascular remodeling. The expression of TLR4, PCNA and NLRP3 inflammasome complex was analyzed by Western blots in the aorta of SHR. RESULTS: We showed that TLR4 overexpression with cDNA enhanced, while knockdown of TLR4 with shRNA inhibited Ang II-induced VSMC proliferation. Besides, TLR4 overexpression upregulated the proteion expression of the NLRP3 inflammasome components including NLRP3, ASC and caspase-1, whereas their corresponding levels of expression were observed to decrease in TLR4 shRNA-transfected VSMCs. Knockdown of TLR4 attenuated vascular remodeling, blood pressure (BP) and the levels of NLRP3, ASC, caspase-1, IL-1ß and IL-18 in SHR aortas. CONCLUSION: This study revealed that TLR4 regulated Ang II-induced VSMC proliferation through modulating the NLRP3 inflammasome. Knockdown of TLR4 attenuated the BP and vascular remodeling by inhibiting the expression of the NLRP3 inflammasome component in SHR. Our results support that TLR4 regulates VSMC proliferation in hypertension via triggering the NLRP3 inflammasome.

MicroRNA-16 directly binds to DEC2 and inactivates the TLR4 signaling pathway to inhibit lupus nephritis-induced kidney tissue hyperplasia and mesangial cell proliferation.

Lupus nephritis (LN) is the most serious manifestation of systemic lupus erythematosus (SLE) and a major risk of mortality. This research focused on the function of microRNA-16 (miR-16) in LN development. Fcgamma receptor II-b-deficient (Fcgr2b-/-) mice with the natural potential to develop SLE- and LN-like diseases were used. Gain- and loss-of-function studies were performed to explore the function of miR-16 in pathological symptoms in mouse kidney tissues and the proliferation of mesangial cells (SV40 MES-13). The putative downstream molecules of miR-16 were explored. Consequently, poor expression of miR-16 was found in kidney tissues. Upregulation of miR-16 inhibited tissue hyperplasia, inflammatory infiltration, glomerular injury and fibrosis but increased cell apoptosis in mouse kidney tissues, and it inhibited proliferation but promoted apoptosis of MES-13 cells as well. miR-16 directly bound to DEC2 and inactivated the TLR4 signaling. DEC2 blocked the protective roles of miR-16 in MES-13 cells. The enhanced proliferation in MES-13 cells following miR-16 inhibition was reversed by chloroquine phosphate, a TLR4 antagonist. To sum up, miR-16 was evidenced to have a potent protective capacity in LN through relieving the LN symptoms in kidney tissues and reducing proliferation of mesangial cells, during which DEC2 silencing and TLR4 signaling deficit were involved.

CircZNF609 is involved in the pathogenesis of focal segmental glomerulosclerosis by sponging miR-615-5p.

As a set of distinct syndromes, focal segmental glomerulosclerosis (FSGS) is the most common cause of adult nephrotic syndrome with diverse mechanisms. We recently found that expression of the circular RNA circZNF609 is increased in renal biopsies of lupus nephritis patients. In the present study, we aimed to determine whether circZNF609 participates in the pathogenesis of FSGS in mice given Adriamycin. In FSGS mice, circZNF609 was upregulated while miR-615-5p was downregulated in FSGS mice analyzed by qPCR and fluorescence in situ hybridization (FISH). Expression of podocyte proteins Wilms tumor 1 (WT1) and podocin were decreased, while expression of collagen 1 (COL1) and transforming growth factor-beta1 (TGF-ß1) were increased on Western blotting. Renal circZNF609 levels were positively correlated and miR-615-5p levels were negatively correlated with the degree of podocyte injury and renal fibrosis. Importantly, circZNF609 and miR-615-5p co-localized to glomeruli and tubules on FISH. Perfect match seeds were found between circZNF609 and miR-615-5p and COL1 mRNA, leading us to explore mechanisms of circZNF609 in bovine serum albumin (BSA) stimulating HK-2 cells, which model the toxicity of proteinuria on tubular cells. In vitro studies, circZNF609 increased and miR-615-5p decreased after BSA treatment and were negatively correlated with each other. COL1 and TGF-ß1 were both upregulated and negatively correlated with miR-615-5p. Lastly, circZNF609 expression increased in glomeruli and tubules of FSGS patient renal biopsies. We conclude that circZNF609 may play an important role in FSGS by sponging miR-615-5p.

MicroRNA-183 exerts a protective role in lupus nephritis through blunting the activation of TGF-ß/Smad/TLR3 pathway via reducing Tgfbr1.

PURPOSE: The role of microRNA (miR)-183 has been elucidated in systemic lupus erythematosus, while whether it is also engaged in the lupus nephritis (LN) development remains opaque. The intention of this study is to examine the relevance of miR-183 downregulation in the pathogenesis of LN. METHODS: The expression of miR-183 was first detected in MRL/lpr mice at weeks 8 and 12, followed by the assessment the effects of miR-183 on renal fibrosis and inflammatory response after overexpression or silencing of miR-183 in mice with LN. We further overexpressed or knocked-down miR-183 in human renal glomerular endothelial cells (HRGECs), and detected the expression patterns of inflammatory factors and Vimentin and α-SMA in the cells. Differentially expressed genes in HRGECs overexpressing miR-183 by microarrays were intersected with targeting mRNAs of miR-183 predicted by bioinformatics websites. The effects of transforming growth factor beta receptor 1 (Tgfbr1) and TGF-ß/Smad/TLR3 pathway on renal damage in mice were verified by rescue experiments. RESULTS: miR-183 expression was notably lower in MRL/lpr mice, and increased miR-183 expression inhibited renal fibrosis and inflammatory response in mice with LN. Moreover, miR-183 inhibitor in HRGECs remarkably promoted the expression of Vimentin and α-SMA and the secretion of inflammatory factors. miR-183 protected the mouse kidney from pathological damages by targeting and inhibiting Tgfbr1 expression. CONCLUSION: miR-183 inhibited the expression of Tgfbr1 by direct targeting to disrupt the TGF-ß/Smad/TLR3 pathway, thus repressing renal fibrosis and the secretion of inflammatory factors in LN.

SNHG6 modulates oxidized low-density lipoprotein-induced endothelial cells injury through miR-135a-5p/ROCK in atherosclerosis.

BACKGROUND: Plenty of long non-coding RNAs (lncRNAs) play vital roles in the progression of atherosclerosis. Small nucleolar RNA host gene 6 (SNHG6) is a well known lncRNA that is aberrantly high expressed in atherosclerosis patients. However, its function and basic mechanism in atherosclerosis events have not been well clarified. METHODS: The expression patterns of SNHG6, miR-135a-5p, ROCK1 and ROCK2 in clinical samples and cells were detected by RT-qPCR assays. Cell Counting Kit-8 (CCK-8), flow cytometry assays, ELISA and reactive oxygen species (ROS) and malondialdehyde (MDA) detection, were performed to assess cell viability, apoptosis, inflammation and oxidative stress, respectively. Western blot analysis was carried out to examine the protein levels of Bax, Bcl-2, and SNHG6. Luciferase reporter and RIP assays were used to confirm the true interaction between SNHG6 and miR-135a-5p, or miR-135a-5p and ROCK. RESULTS: The levels of SNHG6, ROCK1 and ROCK2 were notably increased and miR-135a-5p was decreased in atherosclerosis patients and oxidized low-density lipoprotein (ox-LDL)-treated HUVECs. Knockdown of SNHG6 alleviated ox-LDL-induced injury of HUVECs, while this effect was partly reversed by miR-135a-5p inhibitor. Moreover, overexpression of ROCKs aggravated miR-135a-5p-alleviated atherosclerosis cell injury. SNHG6 contributed to ROCK expression through sequestering miR-135a-5p as a molecular sponge. CONCLUSION: SNHG6 functions as a promoter in atherosclerosis events by targeting miR-135a-5p/ROCK axis in ox-LDL-stimulated HUVECs. This finding will help to develop a novel therapeutic strategy for atherosclerosis.

NORAD affects the progression of diabetic nephropathy through targeting miR-520h to upregulate TLR4.

To uncover the role of NORAD in the progression of diabetic nephropathy (DN) and the underlying mechanism. Relative levels of NORAD and TLR4 in db/m mice and db/db mice were tested. Meanwhile, their levels in glomerular mesangial cells undergoing high-level (H-MC group) or low-level (L-MC) glucose treatment were determined. Regulatory effects of NORAD and TLR4 on proliferative ability and apoptosis in SV40-MES-13 cells were assessed. The interaction in the regulatory loop NORAD/miR-520h/TLR4 was verified through dual-luciferase reporter gene assay, determination of subcellular distribution and RIP (RNA Immunoprecipitation) assay. At last, potential role of the regulatory loop NORAD/miR-520h/TLR4 in regulating DN was clarified. NORAD and TLR4 were upregulated in db/db mice and SV40-MES-13 cells in H-MC group. Overexpression of them promoted proliferative ability and inhibited apoptosis in SV40-MES-13 cells. MiR-520h was confirmed to bind NORAD and TLR4. NORAD, miR-520h and TLR4 were mainly distributed in cytoplasm, which were enriched in anti-Ago2. The regulatory loop NORAD/miR-520h/TLR4 has been demonstrated to promote the progression of DN. The regulatory loop NORAD/miR-520h/TLR4 promotes the proliferative ability and inhibits apoptosis in glomerular mesangial cells, thus aggravating the progression of DN.

miR-150 inhibitor ameliorates adriamycin-induced focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the most common cause of adult nephrotic syndrome in USA. Its mechanisms remain unclear and the effective treatment lacks. We previously reported that upregulation of microRNA (miR)-150 in human podocytes increases profibrotic proteins and decreases anti-fibrotic suppressor of cytokine signaling 1 (SOCS1). We aimed to clarify whether miR-150 inhibitor can ameliorate glomerular injury and to identify its corresponding mechanisms in adriamycin-induced FSGS mice. We found that renal miR-150 increased in adriamycin-induced FSGS mice and FAM-labeled locked nucleic acid-anti-miR-150 (LNA-anti-miR-150) was absorbed by the animal kidneys 6 h after subcutaneous injection. The administration of LNA-anti-miR-150 (2 mg/kg BW twice weekly for 6 w) inhibited renal miR-150 levels without systemic toxicity. With renal miR-150 inhibition, proteinuria, hypoalbuminemia, and hyperlipemia were ameliorated in FSGS mice compared to the scrambled LNA. Meanwhile, the elevated profibrotic proteins and proinflammatory cytokines, decreased antifibrotic SOCS1, and the filtration of T cells in FSGS mice were reverted by LNA-anti-miR-150. Finally, we found that miR-150 most located on podocytes in renal biopsies of FSGS patients. We conclude that LNA-anti-miR-150 might be a novel promising therapeutic agent for FSGS. The renal protective mechanisms might be mediated by anti-fibrosis and anti-inflammation as well as reducing infiltration of T cells in the kidney.

Monoclonal gammopathy of renal significance: clinical manifestation, pathogenic characteristic and treatment.

Monoclonal gammopathy of renal significance (MGRS) is a group of renal disorders caused by a monoclonal immunoglobulin (MIg) secreted by a dangerous plasmatic/B-cell clone hyperplasia through MIg deposition or dysfunction of complement pathway, with increasing risk of progress to end stage renal disease (ESRD) and the underlying hematologic malignancy. The combination of renal biopsy, complete laboratory examination and bone marrow biopsy is an indispensable diagnostic tool for MGRS to identify accurately and unequivocally the pathogenic monoclonal MIg and provide guidance to treatment. Treatment of MGRS is composed of conventional therapy, chemotherapy, and stem cell transplantation to target the underlying clone and eliminate the noxious MIg on the basis of clinical data of some retrospective studies and a small amount of prospective trial. In addition, it is worthwhile point out assessment of therapeutic effect is significantly relevant for renal and overall prognosis. Thus, by comprehensively analyzing the clinical manifestations and pathogenic characteristic of MGRS, early recognition and prompt treatment can improve the prognosis and prevent post-translation recurrence with multidisciplinary cooperation.

MicroRNA-96 regulates pancreatic ß cell function under the pathological condition of diabetes mellitus through targeting Foxo1 and Sox6.

To elucidate the potential function of microRNA-96 in protecting pancreatic ß cell function under the pathological condition of T2DM and the underlying mechanism. Relative levels of microRNA-96 and genes associated with ß cell function in the in vivo and in vitro T2DM and obesity models were detected by qRT-PCR. Insulin functions, including fasting blood glucose, plasma insulin, HOMA-IR, HOMA-%b, glucose tolerance and insulin tolerance, were assessed in microRNA-96 KO mice and wild-type mice fed with normal diet or high-fat diet. Downstream targets of microRNA-96 were verified by dual-luciferase reporter gene assay. Finally, regulatory effects of microRNA-96 on proliferation and apoptosis of MIN6 cells were determined. MicroRNA-96 was upregulated in mice fed with high-fat diet, db/db mice, high-level glucose-treated cells, TNF-α-treated cells, pancreatic cells isolated from the obesity and T2DM patients. Increased fasting blood glucose and HOMA-IR, as well as decreased plasma insulin and HOMA-%b were observed in microRNA-96 KO mice. IPGTT and IPITT results indicated that knockout of microRNA-96 led to pancreatic ß cell dysfunction under the pathological condition of T2DM. Dual-luciferase reporter gene assay confirmed that microRNA-96 could bind Foxo1 and Sox6. MicroRNA-96 negatively regulated Foxo1 and Sox6 levels. Moreover, overexpression of microRNA-96 promoted proliferative ability and inhibited apoptosis in MIN6 cells. Relative levels of Pdx1, Nkx6.1, Cyclin D1 and Cyclin E1 were upregulated in MIN6 cells overexpressing microRNA-96. Opposite results were obtained after knockdown of microRNA-96 in MIN6 cells. MicroRNA-96 is upregulated in pancreatic ß cells under the pathological condition of T2DM. Overexpression of microRNA-96 promotes proliferative ability and inhibits apoptosis in pancreatic ß cells through targeting Foxo1 and Sox6.

Vps15 is critical to mediate autophagy in AngII treated HUVECs probably by PDK1/PKC signaling pathway.

AIMS: Vps15 is an important regulator on the activity of class III PI3K in autophagy induction. AngII plays a positive role of autophagy in the early protection of endothelial cells. In this study, the expression of Vps15 was knocked down using the specific shRNA to investigate the effects of Vps15 on cell autophagy, senescence and apoptosis in HUVECs stimulated by AngII. The associated cell signaling pathway was also explored. MATERIALS AND METHODS: MDC staining was applied to show autophagic bodies. Cell senescence was detected using ß-galactosidase staining. Cell apoptosis was examined by flow cytometry using Annexin V-FITC/PI staining. And western blot was used to evaluate the ratio of LC3-II/I and the activation of associated cell signaling pathway. KEY FINDINGS: Cell autophagy induced by AngII was inhibited in HUVECs transfected with Vps15-shRNA, while cell senescence and apoptosis were enhanced. Rescue experiment revealed that cell autophagy was activated after Vps15 reexpression, while cell senescence and apoptosis were inhibited. Moreover, the phosphorylations of PDK1 and PKC substrates were increased after AngII treatment, which were decreased by Vps15 knockdown. Pretreatment of cells with the inhibitor for PDK1 or PKC attenuated cell autophagy after AngII stimulation, yet promoted cell senescence and apoptosis. The phosphorylations of both PDK1 and PKC were inhibited in cells pretreated with PDK1 inhibitor. Only the activation of PKC was inhibited when the inhibitor for pan-PKC was used. SIGNIFICANCE: These results suggested that Vps15 was critical to the protective autophagy in HUVECs induced by AngII, and PDK1/PKC signaling pathway was probably involved.

circHLA-C Plays an Important Role in Lupus Nephritis by Sponging miR-150.

Circular RNAs (circRNAs) participate in the pathogenesis of various diseases by sponging microRNAs (miRs). However, the roles of circRNAs remain unreported in glomerular diseases. We previously reported that miR-150 positively correlated with renal chronicity index in patients with lupus nephritis (LN). We aimed to investigate renal circRNA profiling and the interaction between circRNAs and miR-150 in LN patients. Six renal biopsies from untreated female patients with LN class IV and five normal kidney tissues from urology patients were used for circRNA sequencing. 171 circRNAs with 2-fold differential expression were identified in LN compared with normal control. Ten selected circRNAs were validated by real-time qPCR, and seven circRNAs showed the same significant increases as the sequencing results. circHLA-C positively correlated with proteinuria (R = 0.92, p < 0.01), serum creatinine (R = 0.76, p = 0.08), renal activity index (R = 0.88, p < 0.05), and crescentic glomeruli (R = 0.93, p < 0.01). Renal circHLA-C increased 2.72-fold, and miR-150 decreased 66% in LN compared with normal control (p < 0.05). Bio-informatic analysis predicted miR-150 was regulated by circHLA-C and displayed one perfect match seed between circHLA-C and miR-150. The renal miR-150 showed a tendency of negative correlation with circHLA-C in LN patients. In conclusion, circHLA-C may play an important role in the pathogenesis of lupus nephritis by sponging miR-150.

Protection of endothelial cells against Ang II-induced impairment: Involvement of both PPARα and PPARγ via PI3K/Akt pathway.

The aim of our study is to explore the involvement of PPARα and PPARγ in Ang II-induced endothelial injury. We found that Ang II significantly elevated the oxidative stress in HUVECs, causing apoptosis and cellular impairment in a time-dependent pattern. Activation of either PPARα by docosahexaenoic acid (DHA) or PPARγ by rosiglitazone protected the endothelial cells. Interestingly, a more significant effect was observed when DHA and rosiglitazone were administrated together. Moreover, we found that this protection was mediated through the PI3K/Akt pathway. Our study may help to understand the mechanism of endothelial dysfunction, contributing to the treatment of hypertension and other endothelial-related diseases.