From genomic insights to clinical hope: Targeting NEU1 in IgA nephropathy.

BACKGROUND: IgA Nephropathy (IgAN), the primary form of glomerulonephritis, presents significant clinical challenges due to its obscure pathogenesis and lack of targeted treatments. We conducted a proteome-wide Mendelian randomization (MR) study to identify therapeutic targets for IgAN. METHODS: Utilizing a plasma proteome dataset comprising 4907 blood plasma proteins as the exposure variable, and renal biopsy-confirmed IgAN cases as the outcome, this study employed MR to pinpoint proteins potentially pathogenic to IgAN. The robustness of our findings was affirmed through external dataset validation, reverse causation testing, and Bayesian colocalization analysis. Additionally, we conducted phenotypic scanning and analyzed downstream metabolites to investigate candidate proteins's biological function. RESULTS: In our study, a significant association was identified between an increase in neuraminidase 1 (NEU1) expression and the risk of IgAN. Specifically, a one standard deviation increase in NEU1 expression was associated with an odds ratio of 11.80 for the development of IgAN (95% confidence interval: 4.03-34.54). This association was substantiated across various statistical models and external validations. Colocalization analysis indicated a shared causal variant between NEU1 expression and IgAN. Furthermore, an increased influenza risk associated with NEU1 was observed, supporting the therapeutic potential of NEU1 inhibitors for IgAN. However, our study found no significant role for neuraminic acid-related metabolites in IgAN's development, suggesting an independent pathway for NEU1's influence. CONCLUSION: This study identifies NEU1 as a promising therapeutic target for IgAN, backed by robust genetic evidence. Future research should explore NEU1's therapeutic potential in diverse populations and clinical scenarios, further establishing its role in IgAN.

Sparsentan ameliorates glomerular hypercellularity and inflammatory-gene networks induced by IgA1-IgG immune complexes in a mouse model of IgA nephropathy.

IgA nephropathy (IgAN) is characterized by glomerular deposition of immune complexes (ICs) consisting of IgA1 with O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These ICs induce kidney injury, and in the absence of disease-specific therapy, up to 40% of patients with IgAN progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. Here, we used a model wherein engineered ICs (EICs) formed from human Gd-IgA1 and recombinant human IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. In this model, we assessed the protective effects of sparsentan, a single-molecule dual endothelin angiotensin receptor antagonist (DEARA) versus vehicle on EIC-induced glomerular proliferation and dysregulation of gene expression in the kidney. Oral administration of sparsentan (60 or 120 mg/kg daily) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity. Furthermore, analysis of changes in the whole kidney transcriptome revealed that key inflammatory and proliferative biological genes and pathways that are upregulated in this EIC model of IgAN were markedly reduced by sparsentan, including complement genes, integrin components, members of the mitogen-activated protein kinase family, and Fc receptor elements. Partial overlap between mouse and human differentially expressed genes in IgAN further supported the translational aspect of the immune and inflammatory components from our transcriptional findings. In conclusion, our data indicate that in the mouse model of IgAN, sparsentan targets immune and inflammatory processes leading to protection from mesangial hypercellularity.NEW & NOTEWORTHY The mechanisms by which deposited IgA1 immune complexes cause kidney injury during early phases of IgA nephropathy are poorly understood. We used an animal model we recently developed that involves IgA1-IgG immune complex injections and determined pathways related to the induced mesangioproliferative changes. Treatment with sparsentan, a dual inhibitor of endothelin type A and angiotensin II type 1 receptors, ameliorated the induced mesangioproliferative changes and the associated alterations in the expression of inflammatory genes and networks.

Protective effect of modified Huangqi Chifeng decoction on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway.

OBJECTIVE: To examine the nephroprotective mechanism of modified Huangqi Chifeng decoction (, MHCD) in immunoglobulin A nephropathy (IgAN) rats. METHODS: To establish the IgAN rat model, the bovine serum albumin, lipopolysaccharide, and carbon tetrachloride 4 method was employed. The rats were then randomly assigned to the control, model, telmisartan, and high-, medium-, and low-dose MHCD groups, and were administered the respective treatments via intragastric administration for 8 weeks. The levels of 24-h urinary protein, serum creatinine (CRE), and blood urea nitrogen (BUN) were measured in each group. Pathological alterations were detected. IgA deposition was visualized through the use of immunofluorescence staining. The ultrastructure of the kidney was observed using a transmission electron microscope. The expression levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-ß1 (TGF-ß1) were examined by immunohistochemistry and quantitative polymerase chain reaction. Levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB) P65, were examined by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. RESULTS: The 24-h urine protein level in each group increased significantly at week 6, and worsen from then on. But this process can be reversed by treatments of telmisartan, and high-, medium-, and low-dose of MHCD, and these treatments did not affect renal function. Telmisartan, and high-, and medium-dose of MHCD reduced IgA deposition. Renal histopathology demonstrated the protective effect of high-, medium-, and low-dose of MHCD against kidney injury. The expression levels of MCP-1, IL-6, and TGF-ß1 in kidney tissues were downregulated by low, medium and high doses of MHCD treatment. Additionally, treatment of low, medium and high doses of MHCD decreased the protein and mRNA levels of TLR4, MyD88, and NF-κB. CONCLUSIONS: MHCD exerted nephroprotective effects on IgAN rats, and MHCD regulated the expressions of key targets in TLR4/MyD88/NF-κB signaling pathway, thereby alleviating renal inflammation by inhibiting MCP-1, IL-6 expressions, and ameliorating renal fibrosis by inhibiting TGF-ß1 expression.

Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

IgA nephropathy (IgAN) is caused by deposition of IgA in the glomerular mesangium. The mechanism of selective deposition and production of IgA is unclear; however, we recently identified the involvement of IgA autoantibodies. Here, we show that CBX3 is another self-antigen for IgA in gddY mice, a spontaneous IgAN model, and in IgAN patients. A recombinant antibody derived from gddY mice bound to CBX3 expressed on the mesangial cell surface in vitro and to glomeruli in vivo. An elemental diet and antibiotic treatment decreased the levels of autoantibodies and IgAN symptoms in gddY mice. Serum IgA and the recombinant antibody from gddY mice also bound to oral bacteria of the mice and binding was competed with CBX3. One species of oral bacteria was markedly decreased in elemental diet-fed gddY mice and induced anti-CBX3 antibody in normal mice upon immunization. These data suggest that particular oral bacteria generate immune responses to produce IgA that cross-reacts with mesangial cells to initiate IgAN.

Deciphering roles of protein post-translational modifications in IgA nephropathy progression and potential therapy.

Immunoglobulin A nephropathy (IgAN), one type of glomerulonephritis, displays the accumulation of glycosylated IgA in the mesangium. Studies have demonstrated that both genetics and epigenetics play a pivotal role in the occurrence and progression of IgAN. Post-translational modification (PTM) has been revealed to critically participate in IgAN development and progression because PTM dysregulation results in impaired degradation of proteins that regulate IgAN pathogenesis. A growing number of studies identify that PTMs, including sialylation, o-glycosylation, galactosylation, phosphorylation, ubiquitination and deubiquitination, modulate the initiation and progression of IgAN. Hence, in this review, we discuss the functions and mechanisms of PTMs in regulation of IgAN. Moreover, we outline numerous compounds that govern PTMs and attenuate IgAN progression. Targeting PTMs might be a useful strategy to ameliorate IgAN.

Influence of IgA nephropathy on the progression of pulpitis and apical periodontitis in HIGA mice.

OBJECTIVES: Immunoglobulin (Ig)A nephropathy has been associated with oral infections such as periodontitis, but its pathogenesis is not fully understood; no treatments exist. This study analyzes the influence of IgA nephropathy, an autoimmune disease, on the pathogenesis of pulpitis and apical periodontitis. METHODS: Two groups of mice were used in pulp infection experiments: high serum IgA nephropathy model mice (HIGA) and control mice (BALB/c). Histologic analyses of the pulp and apical periodontal tissues were performed on days 3, 5, 7, 14, and 28 following oral bacterial infection. The dynamics of odontoblasts, apoptotic cells, and IgA expression were analyzed using anti-Nestin, TUNEL, and anti-IgA staining, respectively. RESULTS: Inflammatory cells infiltrated the exposed pulp at day three in both groups and by 14 days, these cells had infiltrated from the pulp to the apical periodontal tissue. The area of necrotic pulp tissue increased significantly in the control group at seven days. Odontoblasts decreased from day three onwards and disappeared by 28 days in both groups. The number of apoptotic cells in the pulp and apical periodontal tissues was significantly higher in the experimental group at day 28. The experimental group exhibited a significant increase in IgA production in the pulp after 14 days. Bone resorption in the apical periodontal tissue was significantly decreased in the experimental group at day 28. CONCLUSIONS: The results of this study suggest that IgA nephropathy may modulate the inflammatory response and sustain long-term biological defense responses in pulpitis and apical periodontitis in HIGA mice.

LPS/TLR4 Pathway Regulates IgA1 Secretion to Induce IgA Nephropathy.

Context: Studies have reported that the incidence and severity of IgA nephropathy (IgAN) are closely related to the imbalance of the intestinal flora. Imbalance of the intestinal flora may cause abnormalities, such as intestinal mucosal immunity or mesenteric B1 lymphocyte subsets. These can lead to an increase in immunoglobulin A (IgA) production and IgA structural changing, which can eventually cause IgA1 deposition in the glomerular mesangial area and nephritis. Objective: The study intended to explore whether the LPS/TLR4 pathway regulates mesenteric B cells, secreting Gd-IgA1 to induce IgA nephropathy. Design: The research team designed an animal study. Setting: The study took place at Department of Nephrology, Minhang Hospital, Fudan University. Animals: The animals were 60 specific pathogen free (SPF) C57BL/6 (B6, H-2b) male mice from that were 6-8 weeks old and weighed 20-25 grams. Intervention: The research team established a mouse model of IgA nephropathy. The team created five groups of mice: (1) the NC group, a normal negative control group without induced nephropathy and with no treatments; (2) the IgA nephropathy (IgAN) group, a positive control group with induced nephropathy and with no treatments; (3) the IgAN+anti-TLR4 group, an intervention group, with induced nephropathy and with a TLR4-antibody (anti-TLR4) treatment; (4) the IgAN+GEC group, an intervention group, with induced nephropathy and with treatment with glutamine enteric-coated capsules (GEC); and (5) the IgAN+anti-TLR4+GEC group, an intervention group, with induced nephropathy and with treatment with anti-TLR4 and GEC. Outcome Measures: The research team collected the blood and urine of all the mice and used an enzyme-linked immunoassay (ELISA) to analyze the levels of blood creatinine, urine protein, and urea nitrogen (BUN). The team also used the ELISA to analyze signal molecules for serum inflammation: interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), cyclooxygenase-2 (COX2), and galactose-deficient IgA1(Gd-IgA1). The team analyzed the distribution and content of IgA+B220+B lymphocytes in the intestinal tissues of all the mice, using tissue immunofluorescence tracking technology, and used hematoxylin-eosin (HE) staining to analyze the pathological damage in the kidney tissue. For analysis of glomerular IgA deposition, the team used a tissue immunofluorescence technique, and for detection of protein expression-toll-like receptor 4 (TLR4), B-cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL)-in mesenteric lymphoid tissues, the team used western blot analysis. Results: For the five groups of mice, the amount or degree of the physiological indicators and inflammatory factors that ELISA detected, the B lymphocytes and IgA sedimentation that immunofluorescence tracing measured, the kidney pathological that HE staining detected, and the expression of immune-related proteins that western blotting measured, all showed a common trend: IgAN group> IgAN+ glomerular endothelial cells (GEC) group> IgAN+anti-TLR4 group> IgAN+anti-TLR4+GEC group> NC group. Conclusions: The TLR4 antibody and GEC for the treatment of the intestinal tract can regulate and repair intestinal function, so that IgAN can also be relieved at the same time. The results supported the hypothesis that a relationship exists between IgAN and the LPS/TLR4 pathway that regulates mesenteric B cells to secrete low-glycosylated poly-IgA1, which provides a new potential therapeutic plan for IgA nephritis.

Hydroxychloroquine ameliorates immune functionality and intestinal flora disorders of IgA nephropathy by inhibition of C1GALT1/Cosmc pathway.

BACKGROUND: Hydroxychloroquine (HCQ) has emerged as a potential and secure antiproteinuric agent in IgA nephropathy (IgAN). This study endeavored to explore the impact of HCQ on the immune functionality and intestinal flora disorders in IgAN rats, as well as to elucidate the underlying mechanisms through in vivo and in vitro experiments. METHODS: IgAN model was established in Sprague-Dawley rats through the administration of BSA, LPS, and CCl4, and the IgAN rats received a continuous 8-week treatment with HCQ. Moreover, the human glomerular mesangial cells (HMCs) were incubated with IgA1 to establish an in vitro cellular model of IgAN. At the end of experimental period, samples were collected for further analysis. RESULTS: HCQ ameliorated the elevated levels of 24hUTP, SCr, BUN, the number of urinary RBC, and the activation of inflammation-related proteins within the TLR4/NF-κB signaling pathway. In the IgAN rat group, there was a pronounced escalation in IgA deposition, mesangial matrix hyperplasia, and glomerular inflammatory cell infiltration, while the administration of HCQ effectively mitigated these pathological changes. In addition, the reduced production of CD4+CD25+Foxp3+ Treg in the IgAN group was effectively reversed by HCQ. Furthermore, HCQ has the capacity to restore the compromised state of the intestinal mucosal barrier induced by IgAN and mitigate the circumstances of intestinal permeability and disruption in the intestinal flora. CONCLUSION: HCQ diminishes IgA aberrant glycosylation levels, ameliorates renal and intestinal histopathological damage, and attenuates intestinal flora disorders and immune dysfunction in IgAN rats by means of activating the C1GALT1/Cosmc pathway.

EIF2α/ATF4 pathway enhances proliferation of mesangial cell via cyclin D1 during endoplasmic reticulum stress in IgA nephropathy.

IgA nephropathy (IgAN) is an essential cause of kidney failure and end-stage kidney disease worldwide. Mesangial hypercellularity is an important characteristic of IgAN, but the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress is a series of stress responses to restore the function of endoplasmic reticulum. We aimed to explore how ER stress functioned in kidneys of IgAN. We first examined ER stress in IgAN kidneys in vivo and in vitro, by testing the levels of ER stress associated proteins (BIP, p-eIF2α and ATF4). Our results showed that ER stress was activated in IgAN patients, mice and cell model. ER stress activation was related to the distribution of IgA deposition and the degree of mesangial proliferation. To determine the role of ER stress in mesangial cell (MC) proliferation of IgAN, we then tested the levels of ER stress and MC proliferation (cyclin D1, cell viability and cell cycle) through inhibiting ER stress associated proteins. After inhibiting ER stress associated proteins, ER stress was inactivated and cell proliferation was inhibited in MCs. We also explored the correlation between ER stress in the glomerulus and the clinical outcomes of IgAN patients in a prospective study. Patients with lower expression of p-eIF2α or ATF4 had higher rates of hematuria remission, proteinuria remission and clinical remission. In summary, our work outlines that in IgAN, ER stress mediated by eIF2α/ATF4 pathway promotes MC proliferation via up-regulating the expression of cyclin D1. Furthermore, p-eIF2α and ATF4 in the glomerulus negatively correlate with the clinical remission of IgAN patients.

RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

Transglutaminase 2 (TGase2) has been shown to contribute to the mesangial IgA1 deposition in a humanized mouse model of IgA nephropathy (IgAN), but the mechanism is not fully understood. In this study, we found that inhibition of TGase2 activity could dramatically decrease the amount of polymeric IgA1 (pIgA1) isolated from patients with IgAN that interacts with human mesangial cells (HMC). TGase2 was expressed both in the cytosol and on the membrane of HMC. Upon treatment with pIgA1, there were more TGase2 recruited to the membrane. Using a cell model of mesangial deposition of pIgA1, we identified 253 potential TGase2-associated proteins in the cytosolic fraction and observed a higher concentration of cellular vesicles and increased expression of Ras homolog family member A (RhoA) in HMC after pIgA1 stimulation. Both the amount of pIgA1 deposited on HMC and membrane TGase2 level were decreased by inhibition of the vesicle trafficking pathway. Mechanistically, TGase2 was found to be coprecipitated with RhoA in the cellular vesicles. Membrane TGase2 expression was greatly increased by overexpression of RhoA, while it was reduced by knockdown of RhoA. Our in vitro approach demonstrated that TGase2 was transported from the cytosol to the membrane through a RhoA-mediated vesicle-trafficking pathway that can facilitate pIgA1 interaction with mesangium in IgAN.

[Notoginsenoside R_1 interferes with renal oxidative stress and Nrf2/HO-1 signaling pathway to alleviate kidney injury in mice with IgA nephropathy and its mechanism].

The purpose of this study was to investigate the effect of notoginsenoside R_1(NGR_1) on alleviating kidney injury by regulating renal oxidative stress and the Nrf2/HO-1 signaling pathway in mice with IgA nephropathy(IgAN) and its mechanism. The mouse model of IgAN was established using a variety of techniques, including continuous bovine serum albumin(BSA) gavage, subcutaneous injections of carbon tetrachloride(CCl_4) castor oil, and tail vein injections of lipopolysaccharide(LPS). After successful modeling, mice with IgAN were randomly separated into a model group, low, medium, and high-dose NGR_1 groups, and a losartan group, and C57BL6 mice were utilized as normal controls. The model and normal groups were given phosphate buffered saline(PBS) by gavage, the NGR_1 groups were given varying dosages of NGR_1 by gavage, and the losartan group was given losartan by gavage for 4 weeks. The 24-hour urine of mice was collected after the last administration, and serum and kidney tissues of mice were taken at the end of the animal experiment. Then urine red blood cell count(URBCC), 24-hour urine protein(24 h protein), serum creatinine(Scr), and blood urea nitrogen(BUN) levels were measured. The enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of galactose-deficient IgA1(Gd-IgA1), kidney injury molecule 1(Kim-1), and neutropil gelatinase-associated lipocalin(NGAL) in the mouse serum. The assay kits were used to detect the levels of malondialdehyde(MDA) and superoxide dismutase(SOD), and immunofluorescence(IF) was used to detect the expression level of glutathione peroxidase 4(GPX4) in the mesangial region. Western blot was used to detect the protein expression of nuclear transcription factor E2 related factor 2(Nrf2)/heme oxygenase 1(HO-1) signaling pathway in the renal tissue. Hematoxylin-eosin(HE) staining was used to observe pathological alterations in the glomerulus of mice. The results revealed that, as compared with the model group, the serum Gd-IgA1 level, URBCC, 24 h protein level, renal damage markers(Kim-1 and NGAL) in the high-dose NGR_1 group decreased obviously and renal function indicators(BUN, Scr) improved significantly. The activity of SOD activity and expression level of GPX4 increased significantly in the high-dose NGR_1 group, whereas the expression level of MDA reduced and protein expression levels of Nrf2 and HO-1 increased. Simultaneously, HE staining of the renal tissue indicated that glomerular damage was greatly decreased in the high-dose NGR_1 group. In conclusion, this study has clarified that NGR_1 may alleviate the kidney injury of mice with IgAN by activating the Nrf2/HO-1 signaling pathway, improving antioxidant capacity, and reducing the level of renal oxidative stress.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

Network Pharmacology and Experimental Validation to Explore That Celastrol Targeting PTEN is the Potential Mechanism of Tripterygium wilfordii (Lév.) Hutch Against IgA Nephropathy.

Purpose: Accumulating clinical evidence showed that Tripterygium hypoglaucum (Lév.) Hutch (THH) is effective against IgA nephropathy (IgAN), but the mechanism is still unclear. This study is to evaluate the renal protective effect and molecular mechanism of THH against IgAN via network pharmacology, molecular docking strategy and experimental validation. Methods: Several databases were used for obtaining the active ingredients of THH, the corresponding targets, as well as the IgAN-related genes. The critical active ingredients, functional pathways, and potential for the combination of the hub genes and their corresponding active components were determined through bioinformatics analysis and molecular docking. The IgAN mouse model was treated with celastrol (1 mg/kg/d) for 21 days, and the aggregated IgA1-induced human mesangial cell (HMC) was treated with various concentrations of celastrol (25, 50 or 75 nM) for 48 h. The immunohistochemistry and Western blot techniques were applied to evaluate the protein expression of the predicted target. The cell counting kit 8 (CCK8) was used to detect HMC proliferation. Results: A total of 17 active ingredients from THH were screened, covering 165 IgAN-related targets. The PPI network identified ten hub targets, including PTEN. The binding affinity between the celastrol and PTEN was the highest (-8.69 kJ/mol). The immunohistochemistry showed that celastrol promoted the expression of PTEN in the glomerulus of IgAN mice. Furthermore, the Western blot techniques showed that celastrol significantly elevated the expression of PTEN and inhibited PCNA and Cyclin D1 in vitro and in vivo. The CCK8 assay determined that celastrol decreased HMC proliferation in a concentration-dependent manner. Conclusion: This study suggests that activating PTEN by celastrol may play a pivotal role in THH alleviating IgAN renal injury.

Blood TGF-ß1 and miRNA-21-5p levels predict renal fibrosis and outcome in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN), the most common primary glomerulonephritis, often presents as advanced renal failure with end-stage renal disease at diagnosis. Tubulointerstitial injury and fibrosis on histology are the most important predictors of renal outcome. A non-invasive biomarker is required for assessment of progression in IgA nephropathy. We investigated the utility of blood profibrotic molecules, TGF-ß1 and miRNA-21-5p (miR-21), to identify a non-invasive biomarker for renal fibrosis in IgAN. MATERIALS AND METHODS: The study included 30 IgAN (mean age 31.5 ± 9 years) at the time of initial diagnosis, 25 age-sex-matched healthy controls and 10 Lupus nephritis patients as disease controls. Serum TGF-ß1 was analyzed by enzyme-linked immunosorbent assay and plasma miR-21 by qRT-PCR, normalized with U6-snRNA. The levels were correlated with clinical features, laboratory parameters, histological Oxford MEST-C score and renal outcome. RESULTS: The serum TGF-ß1 and plasma miR-21 were significantly higher in patients with IgAN than in healthy controls. TGF-ß1 significantly correlated with serum creatinine, eGFR, Oxford T score and miR-21. High plasma miR-21 was significantly associated with T score and interstitial inflammation. On multivariate analysis, high levels of TGF-ß1 and miR-21 correlated with lower eGFR and T score, respectively. On a follow-up period of 21.5 months, high miR-21 expression at diagnosis was associated (p = 0.02) with a poor renal outcome having a shorter time to doubling of serum creatinine. CONCLUSION: High blood TGF-ß1 and miR-21 expression at diagnosis of IgAN show significant correlation with renal function and degree of chronic tubulointerstitial injury on histology.

Targeted Metabolomics Study of Human Plasma Revealed Activation of the Cytochrome P450 Epoxygenase/Epoxide Hydrolase Axis in Patients with IgA Nephropathy.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis and a leading cause of chronic kidney disease. The pathogenic mechanism of IgAN remains largely unknown and thus a specific therapeutic target is lacking. Here, we reported that the cytochrome P450 (CYP) epoxygenase/epoxide hydrolase (EH) axis was activated in the patients and is likely a therapeutic target for IgAN. Specifically, quantitative profiling of the plasma from IgAN patients and healthy controls revealed significant changes in plasma levels of CYP/EH-mediated lipid epoxides and diols. Subsequently, CYP2C8, CYP2C18, CYP2J2, EPHX1, and EPHX2 were found to be significantly increased in whole blood cells at mRNA levels from the IgAN patients when compared with those of healthy controls. Immunohistochemical analysis showed that all five CYPs and two EHs were upregulated in the kidney tissue from IgAN patients when compared with normative renal tissue, but the expression locations of the proteins were different with most of them. Treatment of HK-2 cells with IgA1 increased cell viability, compressed cell apoptosis, and increased the protein levels of CYP2C9, EPHX1, and EPHX2. All the results agreed that CYPs/EHs axis is likely the prophylactic and therapeutic target for IgAN, providing IgAN patients with a new intervention strategy.

Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese.

Activation of the alternative pathway (AP) of complement is thought to play an important role in Immunoglobin A nephropathy (IgAN). Our previous study showed that rs4151657 within the complement factor B (CFB) gene increased the risk of IgAN. The protein encoded by the CFB gene is an initial factor that promotes AP activation. The aim of this study was to investigate whether other variants of CFB confer susceptibility to IgAN and elucidate their potential roles in AP activation. A total of 1,350 patients with IgAN and 1,420 healthy controls were enrolled and five tag single-nucleotide polymorphisms were selected for genotyping. The levels of key AP components, such as CFB, complement factor H and complement split product C3a, were measured by enzyme-linked immunosorbent assay. Molecular docking and molecular dynamic simulation were carried out to characterize the mutation of residues in the protein structure and the dynamic properties of wide type and mutation models of CFB protein. The allele-specific effect on CFB expression and its binding affinity to C3b were investigated through cell transfection and surface plasmon resonance analysis, respectively. We found that rs12614 significantly reduced the risk of IgAN (OR = 0.69, 95% CI = 0.52-0.91, P = 0.009), and the rs12614-T (R32W mutation) was correlated with lower CFB levels, higher serum C3 level, and less mesangial C3 deposition in patients with IgAN. The structural model showed that the R32W mutation reduced the structural stability of CFB protein. Furthermore, in vitro study revealed that rs12614-T decreased the expression of CFB and reduced its binding affinity to C3b by four-fold compared with rs12614-C. In conclusion, the rs12614-T in CFB was associated with low risk of IgAN probably by attenuating AP activation.

Fucose as a potential therapeutic molecule against the immune-mediated inflammation in IgA nepharopathy: An unrevealed link.

Background: IgA nephropathy (IgAN) is an autoimmune disease that affects people of any age and is an important cause of end-stage renal disease. However, the pathogenesis and pathophysiology of IgAN is not clear. This article aimed to explore the immune-mediated inflammation and genetic mechanisms in IgAN. Methods: The transcriptome sequencing data of IgAN glomeruli in the Gene Expression Omnibus database were downloaded. Single-sample gene set enrichment analysis was used to estimate the immune microenvironment of the merged microarray data and GSE141295. IgAN samples were divided into two clusters by cluster analysis. "limma" and "DEseq2" package in R were used to identify differentially expressed genes (DEGs). The weighted gene co-expression network analysis (WGCNA) was used to identify the co-expression modules related to inflammation in IgAN. R software package "clusterProfiler" was used for enrichment analysis, whereas Short Time-Series Expression Miner (STEM) analysis was used to identify the trend of gene expression. Machine-learn (ML) was performed using the shiny app. Finally, Drug Signatures Database (DSigDB) was used to identify potential molecules for treating IgAN. Results: The infiltration of macrophages in IgAN glomeruli was increased, whereas CD4+ T cells, especially inducedregulatory T cells (iTregs) were decreased. A total of 1,104 common DEGs were identified from the merged data and GSE141295. Brown module was identified to have the highest inflammatory correlation with IgAN using WGCNA, and 15 hub genes were screened from this module. Among these 15 hub genes, 14 increased with the severity of IgAN inflammation based on STEM analysis. Neural network (nnet) is considered as the best model to predict the severity of IgAN. Fucose identified from DSigDB has a potential biological activity to treat IgAN. Conclusion: The increase of macrophages and the decrease of iTregs in glomeruli represent the immune-mediated inflammation of IgAN, and fucose may be a potential therapeutic molecule against IgAN because it affects genes involved in the severe inflammation of IgAN.

Significance of Mannan Binding Lectin-Associated Serine Protease 2 in Urinary Extracellular Vesicles in IgA Nephropathy.

PURPOSE: Immunoglobulin A (IgA) nephropathy (IgAN) is a common chronic glomerulonephritis and the main cause of end-stage renal diseases. Recent evidence suggests that mannan binding lectin associated serine proteases 2 (MASP2) is related to IgAN; therefore, we investigated the expression and significance of MASP2 in serum and urinary extracellular vesicles (UEVs) in patients with IgAN. METHODS: Thirty-eight patients with IgAN and 17 healthy controls were enrolled in this study. UEVs were extracted by ultracentrifugation. The separation by ultra-high-speed centrifuge was verified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Candidate internal references (TSG101, CD9, flotillin, ß-actin and GAPDH) were identified by western blotting in the control group, and the expression of MASP2 in the UEVs was compared. The levels of MASP2 in the serum and UEVs in the IgAN and control groups were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: TEM and NTA results demonstrated that UEVs were successfully extracted. Western blotting results confirmed that TSG101 was suitable as an internal reference for this study. Compared with the control group, the IgAN group showed positive expression of MASP2. MASP2 levels in the UEVs, determined by ELISA, showed significant differences between IgAN and control groups, which were significantly positively correlated with the level of urinary microalbumin. CONCLUSIONS: The level of MASP2 in UEVs was related to IgAN and shows promise as a biomarker for evaluating the severity of renal injury and prognosis of IgAN, thereby helping to elucidate the role of MASP2 in the mannan-binding lectin pathway.

Downregulation of PPARα mediates FABP1 expression, contributing to IgA nephropathy by stimulating ferroptosis in human mesangial cells.

Immunoglobulin A nephropathy (IgAN) is the commonest primary glomerulonephritis, and a major cause of end-stage renal disease; however, its pathogenesis requires elucidation. Here, a hub gene, FABP1, and signaling pathway, PPARα, were selected as key in IgAN pathogenesis by combined weighted gene correlation network analysis of clinical traits and identification of differentially expressed genes from three datasets. FABP1 and PPARα levels were lower in IgAN than control kidney, and linearly positively correlated with one another, while FABP1 levels were negatively correlated with urinary albumin-to-creatinine ratio, and GPX4 levels were significantly decreased in IgAN. In human mesangial cells (HMCs), PPARα and FABP1 levels were significantly decreased after Gd-IgA1 stimulation and mitochondria appeared structurally damaged, while reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly increased, and glutathione and GPX4 decreased, relative to controls. GPX4 levels were decreased, and those of ACSL4 increased on siPPARα and siFABP1 siRNA treatment. In PPARα lentivirus-transfected HMCs stimulated by Gd-IgA1, ROS, MDA, and ACSL4 were decreased; glutathione and GPX4, and immunofluorescence colocalization of PPARα and GPX4, increased; and damaged mitochondria reduced. Hence, PPARα pathway downregulation can reduce FABP1 expression, affecting GPX4 and ACSL4 levels, causing HMC ferroptosis, and contributing to IgAN pathogenesis.

Positive association of serum FUT8 activity with renal tubulointerstitial injury in IgA nephropathy patients.

BACKGROUND: α-1,6 Fucosyltransferase (FUT8) appears to play an essential role in the pathogenesis of renal fibrosis. However, it remained unknown whether FUT8 also contributed to renal fibrosis in immunoglobulin A nephropathy (IgAN). In the present study, we explored the association of serum FUT8 activity with renal tubulointerstitial injury in IgAN patients. METHODS: Serum FUT8 activity was measured in 135 IgAN patients and 68 healthy controls from January 2016 to December 2018. The relationships of serum FUT8 activity with clinical and pathological features were analyzed. RESULTS: Relative to healthy controls, IgAN patients had significantly higher serum FUT8 activity and upregulation of renal FUT8 protein (p < .05). Among IgAN patients, there was a positive correlation of serum FUT8 activity with renal FUT8 protein expression (p < .05). Multivariable logistic regression analyses showed that serum FUT8 activity was significantly associated with serum creatinine and eGFR (p < .05). Based on a cut-off value determined from ROC curve analysis, we divided IgAN patients into a low serum FUT8 activity group (≤12.2 pmol/h/mL, n = 40) and a high serum FUT8 activity group (>12.2 pmol/h/ml, n = 95). The high serum FUT8 activity group had a higher Oxford T score, increased inflammatory cell infiltration, more severe fibrosis and poor renal function (p < .05). CONCLUSION: Serum FUT8 activity was positive association with renal tubulointerstitial injury in IgAN patients.