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More recent studies suggested that metabolic disorders could contribute to the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). The present work aimed at identifying metabolic reprogramming in the kidney of lupus nephritis via proteomics and investigating the potential regulatory mechanism. The proteomic studies on the renal biopsies revealed that the pentose phosphate pathway (PPP) was significantly enriched in the kidneys of LN patients compared with normal controls (NCs). Immunohistochemical stanning of glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme of PPP, verify the results of proteomics. We found that G6PD was highly expressed in the kidneys of LN patients and correlated with several clinicopathological indices. The univariate Cox regression analysis (HR, 95%CI, 1.877 (1.059-3.328), P = 0.031) and Kaplan-Meier (KM) analysis (P = 0.028) suggested that high G6PD expression in the tubulointerstitial area was a risk factor for worse prognosis. Moreover, the Gene set enrichment analysis (GSEA) demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway ranked first in the kidneys of LN patients with high G6PD expression and G6PD was co-localized with mTORC1 activation in the tubule. Immunoglobulin G (IgG) isolated from LN patients significantly activated the mTORC1 pathway and increased G6PD expression, G6PD activity, NADPH production, NADPH oxidase 2 (NOX2) expression, reactive oxygen species (ROS) production, and cell apoptosis in tubule cells in vitro. The above phenotypes were partially rescued after the addition of rapamycin or knock-down of G6PD. Overall, our study suggested that renal G6PD expression was associated with the overall enhanced disease activity and worse renal prognosis. mTORC1 activation might be involved in IgG-LN-induced tubular damage via PPP.

Objective This study was initiated to evaluate mammalian target of rapamycin (mTOR) activation in renal tissue of LN patients. Methods This retrospective study included 187 LN patients, 20 diabetic nephropathy (DN) patients, 10 minimal change disease (MCD) patients and 10 normal controls (NCs). Seven of 187 LN patients had repeated renal biopsies. mTORC1/2 activation was evaluated by immunohistochemistry and multiplexed immunofluorescence. The association of mTORC1/2 activation with the clinicopathologic indices and prognostic outcomes was analysed among 187 LN patients. Proteomics was performed in renal biopsies of 20 LN patients. Proteomics was employed to comprehensively evaluate the impact of mTOR activation on intrarenal gene expression. Results mTORC1/2 was significantly activated in podocytes, mesangial cells, endothelial cells and tubular epithelial cells of LN patients as compared with those with MCD or NC. The glomerular mTORC1 activation was higher in LN patients compared with DN patients. mTORC1, but not mTORC2, activation strongly correlated with serum albumin, complement C3, proteinuria and the following pathological biomarkers of LN: crescent formation, interstitial inflammation and fibrosis. Moreover, mTORC1 activation was identified as a prognostic marker in LN patients. Bioinformatic analyses of proteomics and immunohistochemical data unveiled increased complement activation, antigen presentation and phagocytosis in LN patients with mTORC1 activation. Conclusion Renal mTORC1 activation could be a biomarker to reveal disease activity and predict clinical prognosis in LN patients.

Objective This study was initiated to evaluate the mammalian target of rapamycin (mTOR) signaling pathway involved in renal endothelial- podocyte crosstalk of lupus nephritis (LN) patients. Methods We compared the kidney protein expression patterns of 10 LN patients with severe endothelial- podocyte injury and 3 ones with moderate endothelial- podocyte injury on formalin-fixed paraffin-embedded kidney tissues using label-free liquid chromatography-mass spectrometry (LC-MS) for quantitative proteomics analysis. GO (Gene Ontology) enrichment analyses were performed based on protein intensity levels of differentially expressed proteins (DEPs) in each patient. Enriched mTOR pathway was selected, and the activation of mTOR complexes in renal biopsied specimens were further verified in 176 LN patients. Results Compared with the moderate group, 230 proteins were up-regulated and 54 proteins were down-regulated in severe group. Further GO enrichment analysis was enriched in the “positive regulation of mTOR signaling” pathway. The glomerular activation of mTORC1 was significantly increased in severe group than that in moderate group (P = 0.034) and it was located in podocytes and glomerular endothelial cells, respectively. The glomerular activation of mTORC1 was positively correlated with endocapillary hypercellularity (r = 0.289, P 1240 nm (P

ObjectiveProteomic approach was applied to identify candidate biomarkers of chronicity in patients with proliferative lupus nephritis (LN), and their clinicopathological significance and prognostic values were investigated.MethodsThis study recruited 10 patients with proliferative LN and 6 normal controls (NCs) with proteomic data to compare protein expression profiles, 58 patients with proliferative LN and 10 NCs to verify proteomic data by immunohistochemistry, and 14 patients with proliferative LN with urine samples to evaluate the urinary expression of the biomarker by western blot assay. The composite endpoints included end-stage renal disease and ≥50% reduction from baseline estimated glomerular filtration rate (eGFR).ResultsProteomics detected 48 proteins upregulated in the group with chronicity index (CI) ≥1 compared with the CI=0 and NC groups. Further pathway analysis was enriched in ‘other glycan degradation’. Neuraminidase 1 (NEU1), the most predominant protein in the pathway of other glycan degradation, was highly expressed in the kidney of patients with proliferative LN and could co-localise with podocyte, mesangial cells, endothelial cells and tubule cells. NEU1 expression in the tubulointerstitium area was significantly higher in the CI ≥1 group compared with the CI=0 and NC groups. Moreover, NEU1 expression was significantly correlated with serum creatinine value, eGFR and CI scores, respectively. Urinary NEU1 excretion in the CI ≥1 group was higher than in the CI=0 group and was also positively correlated with CI scores. Furthermore, the high expression of renal NEU1 was identified as an independent risk factor for renal prognosis by multivariate Cox regression analysis (HR, 6.462 (95% CI 1.025 to 40.732), p=0.047).ConclusionsRenal NEU1 expression was associated with pathological CI scores and renal outcomes in patients with proliferative LN.

Background The renal tubules, which have distant metabolic features and functions in different segments, reabsorb >99% of approximately 180 l of water and 25,000 mmol of Na + daily. Defective metabolism in renal tubules is involved in the pathobiology of kidney diseases. However, the mechanisms underlying the metabolic regulation in renal tubules remain to be defined. Methods We quantitatively compared the proteomes of the isolated proximal tubules (PT) and distal tubules (DT) from C57BL/6 mouse using tandem mass tag (TMT) labeling-based quantitative mass spectrometry. Bioinformatics analysis of the differentially expressed proteins revealed the significant differences between PT and DT in metabolism pathway. We also performed in vitro and in vivo assays to investigate the molecular mechanism underlying the distant metabolic features in PT and DT. Findings We demonstrate that the renal proximal tubule (PT) has high expression of lipid metabolism enzymes, which is transcriptionally upregulated by abundantly expressed PPARα/γ. In contrast, the renal distal tubule (DT) has elevated glycolytic enzyme expression, which is mediated by highly expressed c-Myc. Importantly, PPARγ transcriptionally enhances the protease iRhom2 expression in PT, which suppresses EGF expression and secretion and subsequent EGFR-dependent glycolytic gene expression and glycolysis. PPARγ inhibition reduces iRhom2 expression and increases EGF and GLUT1 expression in PT in mice, resulting in renal tubule hypertrophy, tubulointerstitial fibrosis and damaged kidney functions, which are rescued by 2-deoxy- d -glucose treatment. Interpretation These findings delineate instrumental mechanisms underlying the active lipid metabolism and suppressed glycolysis in PT and active glycolysis in DT and reveal critical roles for PPARs and c-Myc in maintaining renal metabolic homeostasis. FUND: This work was supported by the National Natural Science Foundation of China (grants 81572076 and 81873932; to Q.Z.), the Applied Development Program of the Science and Technology Committee of Chongqing (cstc2014yykfB10003; Q.Z.), the Program of Populace Creativities Workshops of the Science and Technology Committee of Chongqing (Q.Z.), the special demonstration programs for innovation and application of techniques (cstc2018jscx-mszdX0022) from the Science and Technology Committee of Chongqing (Q.Z.).

The cyclic GMP-AMP synthase (cGAS), upon cytosolic DNA stimulation, catalyzes the formation of the second messenger 2′3′-cGAMP, which then binds to stimulator of interferon genes (STING) and activates downstream signaling. It remains to be elucidated how the cGAS enzymatic activity is modulated dynamically. Here, we reported that the ER ubiquitin ligase RNF185 interacted with cGAS during HSV-1 infection. Ectopic-expression or knockdown of RNF185 respectively enhanced or impaired the IRF3-responsive gene expression. Mechanistically, RNF185 specifically catalyzed the K27-linked poly-ubiquitination of cGAS, which promoted its enzymatic activity. Additionally, Systemic Lupus Erythematosus (SLE) patients displayed elevated expression of RNF185 mRNA. Collectively, this study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS, shedding light on the regulation of cGAS activity in innate immune responses., Author summary Ubiquitination has been demonstrated to serve as an effective means to catalyze the rapid, dynamic and versatile regulatory processes that are activated when hosts face microbes. Given the critical functions of cytosolic DNA sensing pathway in anti-viral innate immune responses and the pathogenesis of autoimmune diseases, they are subjected to manifold spatial and temporal modulations shaping the strength and duration of the signaling pathways. Recent progress has characterized the cyclic GMP-AMP synthase (cGAS) as the primary DNA sensor that initiates stimulator of interferon genes (STING)-dependent signaling pathway. However, it remains poorly understood how cGAS activity is modulated dynamically. In this study, we identify E3 ubiquitin ligase RNF185 as a positive regulator of cGAS-STING signaling. Knockdown of RNF185 significantly attenuates IRF3-responsive gene expression. ER-resident RNF185 interacts with cGAS and catalyzes the K27-linked poly-ubiquitination of cGAS upon HSV-1 challenges, which thus potentiates cGAS enzymatic activity. Notably, systemic lupus erythematosus (SLE) patients have elevated expression of RNF185 mRNA. Our study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS and suggests RNF185 as an important target for modulating antiviral response.

Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.

Tetratricopeptide repeat domain 36 (Ttc36), whose coding protein belongs to tetratricopeptide repeat (TPR) motif family, has not been studied extensively. We for the first time showed that Ttc36 is evolutionarily conserved across mammals by bioinformatics. Rabbit anti-mouse Ttc36 polyclonal antibody was generated by injecting synthetic full-length peptides through "antigen intersection" strategy. Subsequently, we characterized Ttc36 expression profile in mouse, showing its expression in liver and kidney both from embryonic day 15.5 (E15.5) until adult, as well as in testis. Immunofluorescence staining showed that Ttc36 is diffusely expressed in liver, however, specifically in kidney cortex. Thus, we further compare Ttc36 with proximal tubules (PT) marker Lotus Tetragonolobus Lectin (LTL) and distal tubules (DT) marker Calbindin-D28k respectively by double immunofluorescence staining. Results showed the co-localization of Ttc36 with LTL rather than Calbindin-D28k. Collectively, on the basis of the expression pattern, Ttc36 is specifically expressed in proximal distal tubules.

Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells.

Accumulating evidence demonstrated that miRNAs are highly involved in kidney fibrosis and Epithelial-Eesenchymal Transition (EMT), however, the mechanisms of miRNAs in kidney fibrosis are poorly understood. In this work, we identified that miR542-3p could promote EMT through down-regulating bone morphogenetic protein 7 (BMP7) expression by targeting BMP7 3′UTR. Firstly, real-time PCR results showed that miR542-3p was significantly up-regulated in kidney fibrosis in vitro and in vivo. Moreover, Western blot results demonstrated that miR542-3p may promote EMT in the NRK52e cell line. In addition, we confirmed that BMP7, which played a crucial role in anti-kidney fibrosis and suppressed the progression of EMT, was a target of miR542-3p through Dual-Luciferase reporter assay, as did Western blot analysis. The effects of miR542-3p on regulating EMT could also be suppressed by transiently overexpressing BMP7 in NRK52e cells. Taken together, miR542-3p may be a critical mediator of the induction of EMT via directly targeting BMP7.

MicroRNAs (miRNAs) possess an important regulating effect among numerous renal diseases, while their functions in the process of epithelial-to-mesenchymal transition (EMT) after podocyte injury remain unclear. The purpose of our study is to identify the potential functions of miR-30a in EMT of podocytes and explore the underlying mechanisms of miR-30a in the impaired podocytes. The results revealed that downregulation of miR-30a in podocyte injury animal models and patients, highly induced the mesenchymal markers of EMT including Collagen I, Fibronectin and Snail. Furthermore, overexpression of miR-30a enhances epithelial markers (E-cadherin) but diminished mesenchymal markers (Collagen I, Fibronectin and Snail) in podocytes. In addition, we established miR-30a target NFATc3, an important transcription factor of Non-canonical Wnt signaling pathway. More importantly, our findings demonstrated that the augmentation of miR-30a level in podocytes inhibits the nuclear translocation of NFATc3 to protect cytoskeleton disorder or rearrangement. In summary, we uncovered the protective function of miR30a targeting NFATc3 in the regulation of podocyte injury response to EMT.

The upregulation of Wnt/β-catenin signaling occurs in virtually all types of kidney disease and is associated with podocyte injury. However, the precise mechanisms involved in the development of kidney disease remain to be elucidated. MicroRNAs (miRNAs or miRs) are a class of short non-coding RNAs and they have been shown to be regulators of gene expression, mainly by binding to the untranslated region (UTR) of mRNAs. The aim of the present study was to determine the role of the 2 members of the miR-135 family (miR-135a and miR-135b) in podocyte injury and to elucidate the mechanisms responsible for the damage to podocytes. The results revealed that miR-135a and miR-135b were upregulated in models of podocyte injury and in glomeruli isolated from patients with focal segmental glomerulosclerosis (FSGS). The ectopic expression of miR-135a and miR-135b led to severe podocyte injury and the disorder of the podocyte cytoskeleton. Our findings demonstrated that miR-135a and miR-135b activated Wnt/β-catenin signaling and induced the nuclear translocation of β-catenin. Using luciferase reporter assays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, glycogen synthase kinase 3β (GSK3β) was identified as a target gene of miR-135a and miR-135b. To the best of our knowledge, this is the first study to demonstrate that members of the miR-135 family (specifically miR-135a and miR-135b) regulate the expression of GSK3β, thus playing a role in the development of podocyte injury and the disorder of the podocyte cytoskeleton. This is an important finding as it may contribute to the development of novel therapeutics for podocyte injury-associated glomerulopathies.

A1CF (apobec-1 complementation factor) acts as a component of the apolipoprotein-B messenger RNA editing complex. Previous researches mainly focused on its post-transcriptional cytidine to uridine RNA editing. However, few study reported its role in progression of breast carcinoma cells. Wound healing assay and flow cytometry were applied to detect the migration and apoptosis; western blot, real-time polymerase chain reaction, and dual-luciferase assays were applied to investigate the potential regulation mechanism of A1CF-mediated cell migration and apoptosis. Knockdown of A1CF decreased cell migration and enhanced cell apoptosis in MCF7 cells in vitro. Western blot analysis showed that knockdown of A1CF decreased Dickkopf1 but increased c-Myc and β-catenin expression, and overexpression of A1CF can get opposite results. Knockdown of Dickkopf1 in A1CF-overexpressed cells decreased cell migration and enhanced cell apoptosis compared with A1CF-overexpressed cells. Luciferase-fused 3′ untranslated region of human Dickkopf1 activity was highly upregulated in A1CF-overexpressed MCF7 cells, but this upregulation can be inhibited by mutating conserved binding motifs of Dickkopf1 3′ untranslated region. A1CF played a crucial role in cell migration and survival through affecting 3′ untranslated region of Dickkopf1 to upregulate its expression in MCF7 cells.

Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.

MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is to identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3′UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3′UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3′UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.

The metanephric mesenchyme (MM) cells are a subset of kidney progenitor cells and play an essential role in mesenchymal-epithelial transition (MET), the key step of nephron generation. Six2, a biological marker related to Wnt signaling pathway, promotes the proliferation, inhibits the apoptosis and maintains the un-differentiation of MM cells. Besides, LiCl is an activator of Wnt signaling pathway. However, the role of LiCl in cellular regulation of MM cells remains unclear, and the relationship between LiCl and Six2 in this process is also little known. Here, we performed EdU assay and flow cytometry assay to, respectively, detect the proliferation and apoptosis of MM cells treated with LiCl of increasing dosages. In addition, reverse transcription-PCR (RT-PCR) and Western-blot were conducted to measure the expression of Six2 and some maker genes of Wnt and bone-morphogenetic-protein (BMP) signaling pathway. Furthermore, luciferase assay was also carried out to detect the transcriptional regulation of Six2. Then we found LiCl promoted MM cell proliferation at low-concentration (10, 20, 30, and 40 mM). The expression of Six2 was dose-dependently increased in low-concentration (10, 20, 30, and 40 mM) at both mRNA and protein level. In addition, both of cell proliferation and Six2 expression in MM cells declined when dosage reached high-concentration (50 mM). However, Six2 knock-down converted the proliferation reduction at 50 mM. Furthermore, Six2 deficiency increased the apoptosis of MM cells, compared with negative control cells at relative LiCl concentration. However, the abnormal rise of apoptosis at 30 mM of LiCl concentration implies that it might be the reduction of GSK3β that increased cell apoptosis. Together, these demonstrate that LiCl can induce the proliferation and apoptosis of MM cells coordinating with Six2.