Virus-associated Glomerulonephritis Research Articles

Exosomal miR-223-3p from bone marrow mesenchymal stem cells targets HDAC2 to downregulate STAT3 phosphorylation to alleviate HBx-induced ferroptosis in podocytes.

Background: Hepatitis B virus associated-glomerulonephritis (HBV-GN) is one of the major secondary renal diseases in China, and microRNAs (miRNAs) in bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) can attenuate HBV-X protein (HBx)-induced ferroptosis in renal podocytes, but the exact mechanism remains unclear. This study aimed to investigate the protective mechanism of miR-223-3p in BMSC-Exo in HBx-induced ferroptosis in podocytes. Methods: The study employed human renal podocyte cells (HPCs), bone marrow-derived mesenchymal stem cells (BMSCs), as well as kidney tissue from C57BL/6 mice and HBx transgenic mice. Initially, the correlation between STAT3 phosphorylation and ferroptosis was authenticated through the administration of signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitors in both in vivo and in vitro settings. Furthermore, the effect of HDAC2 overexpression on STAT3 phosphorylation was examined. Subsequently, the association between BMSC-Exo carrying miR-223-3p, HDAC2, and the phosphorylation of STAT3 in HPCs ferroptosis and injury induced by HBx was assessed. The interaction between miR-223-3p and HDAC2 was confirmed via RNA immunoprecipitation assay. Various techniques such as cell counting kit-8 assay, western blot, RT-qPCR, immunofluorescence, flow cytometry, lipid peroxidation assay kit, iron assay kit, transmission electron microscopy, and hematoxylin-eosin staining were employed to visualize the extent of HBx-induced podocyte injury and ferroptosis in both in vivo and in vitro. Results: The attenuation of podocyte ferroptosis can be achieved by inhibiting the phosphorylation of STAT3 in podocytes induced by HBx. Conversely, the upregulation of HDAC2 can enhance STAT3 phosphorylation, thereby promoting podocyte ferroptosis. MiR-223-3p was capable of directly exerting negative regulation on HDAC2 expression. BMSC-Exo carrying miR-223-3p can effectively suppress the expression of HDAC2, ultimately leading to reduce HBx-induced ferroptosis in podocytes by targeting HDAC2 with miR-223-3p and downregulating STAT3 phosphorylation. Conclusion: This study evidences the potential of BMSC-Exo mediated delivery of miR-223-3p in mitigating HBx-induced ferroptosis in podocytes, thereby offering a novel therapeutic target and approach for treating HBV-GN and alleviating renal injury.

Clinicopathologic features and long-term prognosis of hepatitis B virus-associated glomerulonephritis: a retrospective cohort study

BackgroundHepatitis B virus-associated glomerulonephritis is a common form of secondary glomerulonephritis in China. However, the clinicopathological features and long-term prognosis of Hepatitis B virus-associated Glomerulonephritis remain only partially known.MethodsBiopsy-proven Hepatitis B virus-associated Glomerulonephritis patients were enrolled between November 1994 and December 2013 at our center. The composite endpoints were doubling serum creatinine, end-stage renal disease, or death from renal disease during follow-up. The clinicopathological features and predictors of the long-term prognosis of Hepatitis B virus-associated Glomerulonephritis patients were explored.ResultsThe median age of the 259 Hepatitis B virus-associated Glomerulonephritis patients was 31.0 years (IQR 24.0–40.0), and 71.0% were males. Among the patients, 45.2% presented with nephrotic syndrome, and 45.9% presented with proteinuria combined with hematuria. The two most prevalent pathological patterns were IgA nephropathy (27.0%) and membranous nephropathy (27.0%). The mean follow-up period was 68.8 ± 46.9 months. The 3-, 5-, and 10-year clinical event-free survival rates were 93.4%, 85.2%, and 70.3%, respectively. Multivariable Cox regression analysis showed that hypertension (HR 2.580, 95% CI 1.351–4.927, P = 0.004), hyperuricemia (HR 2.101, 95% CI 1.116–3.954, P = 0.021), glomerulosclerosis (P = 0.001), and intrarenal arterial lesions (P = 0.041) were independent predictors of composite clinical event endpoint. Patients in the antiviral therapy group exhibited a significantly better prognosis compared to those who received no antiviral therapy (log-rank χ2 = 5.772, P = 0.016).ConclusionHepatitis B virus-associated Glomerulonephritis has specific clinicopathologic features and should not be considered a benign disease in adults. Hypertension, hyperuricemia, glomerulosclerosis, and intrarenal arterial lesions were independent predictors of the long-term prognosis in Hepatitis B virus-associated Glomerulonephritis patients. Antiviral therapy could be effective in improving the long-term prognosis of Hepatitis B virus-associated Glomerulonephritis patients.Graphic abstract

Bone marrow mesenchymal stem cell-derived exosomes protect podocytes from HBx-induced ferroptosis.

Hepatitis B virus-associated glomerulonephritis (HBV-GN) is a common secondary kidney disease in China, the pathogenesis of which is not completely clear, and there is still a lack of effective treatment. The mechanism of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) was investigated by using HBx-transfected human renal podocytes. Cell viability was detected by CCK8 assay. Iron and malondialdehyde (MDA) contents were detected by using commercial kits. Reactive oxygen species (ROS) levels were measured by flow cytometry analysis. The expression of ferroptosis related molecules was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The effect of miR-223-3p transferred by BMSC-derived exosomes on HBx-overexpressing podocytes was proved by using miR-223-3p inhibitor. The cell viability of podocytes reduced at 72 h or 96 h after the transfection of lentivirus overexpressing HBx protein (p < 0.05). Ferroptosis-related proteins, including glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) were down-regulated upon HBx overexpression, while acyl-CoA synthetase long-chain family member 4 (ACSL4) was up-regulated (p < 0.05). Intracellular levels of iron, MDA, and ROS were also enhanced (p < 0.05). BMSC-derived exosomes protected against ferroptosis induced by HBx overexpression in podocytes. miR-223-3p was enriched in BMSC-derived exosomes. Application of miR-223-3p inhibitor reversed the protective effect of BMSC-derived exosomes on HBx-induced ferroptosis in podocytes. BMSC-derived exosomes inhibit HBx-induced podocyte ferroptosis by transferring miR-223-3p.

Efficacy and safety of entecavir for hepatitis B virus-associated glomerulonephritis with renal insufficiency.

HBV-GN is one of the most common secondary kidney diseases in China. Entecavir is a first-line antiviral therapy in patients with HBV-GN. This retrospective study explored whether entecavir is effective and safe for the treatment of HBV-GN with renal insufficiency. We screened patients diagnosed with HBV-GN in The Affiliated Hospital of Qingdao University who had elevated serum creatinine levels. Group 1 (30 patients) was given entecavir as antiviral treatment. Group 2 (28 patients) was treated with ARBs. Changes in renal function and the possible influencing factors were observed, with a mean follow-up duration of 36months. At the end of follow-up, the elevation in the serum creatinine level and reduction in the eGFR were greater in group 1 than in group 2. The overall renal survival rate, using eGFR < 15ml/min as the primary renal end point, was 96.7% in group 1 and 67.9% in group 2. Urine protein excretion was decreased in both groups. Treatment with entecavir and the remission of proteinuria were protective factors against renal function impairment, while a lower baseline eGFR was a risk factor for progression to ESRD. Entecavir slows the progression of renal function impairment in HBV-GN and exerts a significant renal protective effect.

HBx-induced PLA2R overexpression mediates podocyte pyroptosis through the ROS-NLRP3 signaling pathway

Introduction Hepatitis B virus-associated glomerulonephritis (HBV-GN) is one of the main types of secondary glomerular diseases, and podocyte injury is an important pathogenic mechanism of HBV-GN, participating in the occurrence and development of HBV-GN. However, the specific mechanism of podocyte injury remains to be studied. Methods Human renal podocytes cultured in vitro were divided into six groups. The podocyte morphology was observed under a transmission electron microscope, and the expression of M-type phospholipase A2 receptor (M-PLA2R) on the podocyte membrane was observed by indirect immunofluorescence staining under a fluorescence microscope. The pyroptosis rate and reactive oxygen species (ROS) of podocytes were assessed by FLICA/PI double staining and flow cytometry. Western blot (WB) and quantitative real-time PCR (qPCR) were used to determine the expression of PLA2R, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β, and IL-18. Results Hepatitis B virus X (HBx) transfected into human renal podocytes in vitro induced the overexpression of PLA2R. Moreover, the overexpressed PLA2R combined with secretory phospholipase A2 group IB (sPLA2-IB) aggravated podocyte injury and increased the pyroptosis rate. In addition, the expression of ROS, the NLRP3 inflammasome and downstream inflammatory factors was increased. In contrast, after inhibiting the expression of PLA2R and ROS, podocyte damage was alleviated, and the pyroptosis rate and the expression of genes related to the ROS-NLRP3 signaling pathway were decreased. Conclusion HBx-induced PLA2R overexpression on the podocyte membrane can significantly upregulate the ROS-NLRP3 signaling pathway, thereby mediating podocyte pyroptosis.

Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

Clinical Effect of Lamivudine Combined with Leflunomide and Methylprednisolone Tablets in the Treatment of Hepatitis B Virus-Associated Glomerulonephritis and Its Influence on Renal Function Indicators.

In this study, the clinical effect of lamivudine combined with leflunomide and methylprednisolone in the treatment of hepatitis B virus-associated glomerulonephritis (HBV-GN) and their influence on renal function indexes was explored. Patients with HBV-GN were selected for retrospective analysis and divided into the group B and group A, with 41 cases in each group. The group B was given leflunomide and methylprednisolone, whereas the group A was supplemented with lamivudine. The level of 24 h proteinuria (PRO), albumin (ALB), beta2-microglobulin (β2-MG), alanine aminotransferase (ALT), interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in two groups was measured. The clinical efficacy, adverse reactions appetite, spirit, sleep and daily life scores of the two groups were recorded. With the extension of treatment time to end of the treatment, the level of 24 h PRO, ALB and β2-MG in the group A significantly changed compared with that before treatment (p < 0.05). Moreover, the level of ALT, IFN-γ and IL-4 in the two groups significantly decreased compared with that before treatment, and the level of the three indexes in the group A decreased more significantly (p < 0.05). The total effective rate in the group A was higher than that in the group B (p < 0.05). The occurrence of adverse reactions showed no statistically significant difference between the two groups. After treatment, scores of appetite, spirit, sleep and daily living were increased in the two groups, and the increase in the group A was more significant than that in the group B (p < 0.05). Lamivudine combined with methylprednisolone and leflunomide treatment is conducive to clearing Hepatitis B virus (HBV) and improving renal function.

Role of fluid-phase complement system regulation in the development of hepatitis C virus-associated glomerulonephritis.

ObjectivesIt is not known why only some hepatitis C virus (HCV) infected patients develop glomerulonephritis (GN). Therefore, we investigated the role of soluble complement regulators in the development of HCV associated GN.MethodsPatients with HCV associated GN who were admitted to our nephrology unit between July 2016 and July 2018 were recruited to the study (group 1). Two other age and sex matched groups were studied as control groups: patients with HCV without GN (group 2) and healthy HCV negative volunteers (group 3). There were 26 participants in each of the three groups at the end of the recruitment period. An assay of serum fluid-phase complement regulators was performed using enzyme linked immunosorbent assay technique. Three complement single nucleotide polymorphisms (SNPs) were analyzed using real time polymerase chain reaction (Taqman; thermo fisher scientific): rs2230199 and rs1047286 for complement 3 (C3) and rs800292 for complement factor H (CFH).ResultsSerum levels of complement 4 binding protein (C4BP) were significantly lower in group 1 (median 70 ng/ml) than in groups 2 (median 88.8 ng/ml) and 3 (median 82.8 ng/ml) with p value of 0.007. The minor allele (allele A) of rs800292 for CFH was significantly higher in group 2 and group 3 (G 54% and A 46%) than in group 1 (G 73% and A 27%), p = 0.04.ConclusionsLow C4BP levels are associated with GN in HCV infected patients. In addition, rs800292 SNP in CFH protects against GN in patients with HCV.

DNA methyltransferase 1 knockdown reverses PTEN and VDR by mediating demethylation of promoter and protects against renal injuries in hepatitis B virus-associated glomerulonephritis

BackgroundAberrant DNA methylation patterns, including hypermethylation of key genes that inhibit fibrosis and inflammation, have been described in human kidney diseases. However, the role of DNA methyltransferase 1 (DNMT1) in hepatitis B virus-associated glomerulonephritis (HBV-GN) remains unclear.MethodsWe explored the underlying mechanism by establishing HBV X protein (HBx) overexpressing renal tubular epithelial (HK-2) cells and human podocytes with DNMT1 knockdown. Using RNA-sequencing to determine the downstream targets of DNMT1 and evaluate its levels of promoter methylation. HBV transgenic mice were used to examine the effects of DNMT1 inhibitor on renal in vivo.ResultsDNMT1 was significantly upregulated in the renal tissue of HBV-GN patients, accompanied by injuries of HK-2 cells and podocytes. HBx markedly upregulated DNMT1 and induced epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells and human podocytes. This increased DNMT1 expression was attenuated after DNMT1 knockdown, accompanied by restored HK-2 cells and podocyte injuries resulting from the activation of PI3K/Akt/mTOR and nuclear factor-kappa B (NF-κB) pathways. Hypermethylation of the phosphatase and tensin homolog (PTEN) promoter and vitamin D receptor (VDR) was induced in HBx-overexpressing HK-2 cells and podocytes, respectively, whereas DNMT1 knockdown effectively corrected these alterations. Furthermore, PTEN and VDR ablation resulted in marked EMT and inflammation induction in HBx-overexpressing HK-2 cells and human podocytes even with DNMT1 knockdown. Downregulation of the PI3K/Akt/mTOR-related pathway attenuated HBx-induced EMT and inflammation in HK-2 cells. Luciferase reporter assay revealed VDR as a direct target of the Snail family transcriptional repressor 1 (SNAI1) in HBx-overexpressing podocytes. DNA methylation inhibitor 5-azacytidine alleviated urinary protein and renal inflammation in HBV transgenic mice via PTEN-PI3K/Akt signaling and VDR signaling axis.ConclusionsOur study clarifies the potential epigenetic mechanisms underlying HBx-induced renal injuries in HBV-GN and the renoprotective effects of inhibiting DNMT1, which can provide important insights into the development of treatments for HBV-GN.

The efficacy and safety of tacrolimus and entecavir combination therapy in the treatment of hepatitis B virus-associated glomerulonephritis: a multi-center, placebo controlled, and single-blind randomized trial.

The proteinuria remission in hepatitis B virus-associated glomerulonephritis (HBV-GN) patients with massive proteinuria treated with antiviral therapy was low. Tacrolimus (TAC) is effective in primary nephropathy and can inhibit HBV infection by inhibiting HBV binding to sodium taurocholate cotransporting polypeptide on liver cells. This study evaluated the efficacy and safety of TAC combined with ETV compared with entecavir (ETV) monotherapy in HBV-GN. Patients diagnosed with HBV-GN were recruited for this prospective, randomized, controlled, multicenter, single-blinded study in China. Patients were given TAC and ETV therapy (the TAC+ETV group) or placebo and ETV therapy (the ETV group) for 26 weeks. The efficacy endpoints included proteinuria remission, including complete and partial remission (CR and PR), the change of 24-hour proteinuria (24 h UP) and HBV DNA titer. The safety endpoints were the incidence of HBV virologic breakthrough and adverse events. There were 14 patients in the TAC+ETV group and 17 patients in the ETV group. In the intention-to-treat analyses, 64.3% (9/14) of patients in the TAC+ETV group and 58.8% (10/17) in the ETV group achieved PR or CR at 26 weeks (P=0.38). At week 14, 42.9% (6/14) and 41.2% (7/17) of patients in the TAC+ETV group and the ETV group, respectively, achieved PR or CR (P=0.23). At week 26, the 24 h UP had decreased by 2.63±6.33 g from baseline in the TAC+ETV group and 1.42±4.34 g in the ETV group (P=0.55). The serum albumin increased by 11.1±7.30 g/L from baseline in the TAC+ETV group and 3.81±5.09 g/L in the ETV group (P<0.001). Log10 HBV DNA decreased by 1.49±2.04 from baseline in the TAC+ETV group and 2.47±2.08 in the ETV group (P=0.37); 28.6% (4/14) patients had HBV DNA virologic breakthrough in the ETV group, while none in the TAC+ETV group (P=0.29). In adult HBV-GN patients, TAC and ETV combination therapy may significantly improve serum albumin levels without increasing the risk of HBV reactivation compared with entecavir monotherapy. ClinicalTrials.gov Identifier NCT03062813.

IFI16 induces inflammation in hepatitis B virus-associated glomerulonephritis by regulating the Caspase-1/ IL-1 \xdf pathway

Aims and backgroundIFI16 plays an important role in innate immunity against invasive microbial infection by sensing double-stranded DNA viruses due to caspase-1-dependent inflammasome activation and subsequent maturation and secretion of IL-1β. However, the role of IFI16 in regulating the immune response to viruses in Hepatitis B Virus-Associated Glomerulonephritis (HBV-GN), especially in sensing hepatitis B virus (HBV), has not been determined. In this study, we investigated the inflammatory role of IFI16 in HBV-GN.MethodsA total 75 kidney tissue including 50 HBV-GN and 25 chronic glomerulonephritis (CCN) were collected to determine the expression of IFI16, Caspase-1 and IL-1β using immunohistochemistry (IHC), then the correlation between them was analyzed. In vitro, the primary human glomerular mesangial (HGM) cells and HEK-293 T cell lines were used in this study. The cell lines were both co-transfected with HBVDNA and overexpression or silencing IFI16. Quantitative Real-time PCR and western blotting were used to determine the expression of IFI16, Caspase-1 and IL-1β.ResultsIFI16 expression in HBV-GN biopsies (80.0%) was significantly higher than in CGN (24.0%) and positively correlated with HBVDNA,caspase-1 and IL-1β expression in HBV-GN. Meanwhile, over expression of IFI16 increased caspase-1 and IL-1β expression in HBV-infected HGM and HEK-293 T cell lines, knockdown of IFI16 mRNA by siRNA resulted in downregulation of the caspase-1 and IL-1β expression in both cell lines.ConclusionsThe elevation of IFI16 during HBV infection or replication may contribute to renal damage due to inflammation, thus providing a putative therapeutic target and a new avenue for researching the pathogenesis of HBV-GN.

Hepatitis B virus X mediates podocyte pyroptosis by regulating the ROS/NLRP3 signaling pathway in hepatitis B virus-associated glomerulonephritis

Objective(s):This study was designed to investigate whether HBx-induced podocyte injury is related to the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and the specific mechanism of the oxidative stress pathway in hepatitis B virus-associated glomerulonephritis (HBV-GN).Materials and Methods:The HBx gene was overexpressed in renal podocytes to mimic HBV-GN. Podocyte morphology was observed under a scanning electron microscope. Reactive oxygen species (ROS) generation was detected by dichlorodihydrofluorescein diacetate (DCFH-DA) assay. The podocytes in each group were treated with Hoechst 33342 and subjected to immunofluorescence staining. Caspase-1 activity and LDH levels were assessed with a Caspase-1 Activity Assay Kit and an LDH ELISA Kit, respectively. The expression of all pyroptosis-related proteins was examined by Western blot analysis.Results:Pyroptosis-related proteins, including NLRP3, apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β, and IL-18 (P<0.05), were up-regulated upon HBx overexpression, and caspase-1 enzyme activity and LDH and Desmin expression were also enhanced (P<0.05). NLRP3 knockdown attenuated the increased expression of pyroptosis-related proteins upon HBx overexpression (P<0.05), which was also achieved by the addition of an ROS inhibitor (P<0.05).Conclusion:HBx regulates podocyte pyroptosis in HBV-GN by targeting the NLRP3 inflammasome, and mitochondrial oxidative stress plays an important role in this process.

Hepatitis B Virus-Related Glomerulonephritis with Positive and Negative Serum HBsAg: Different Clinicopathologic Characteristics of Two Clinical Subtypes.

IntroductionThe clinicopathologic characteristics of Hepatitis B virus-associated glomerulonephritis (HBV-GN) patients with different serum HBsAg are not well known. This study aims to investigate the characteristics and treatments between HBV-GN patients with positive and negative serum HBsAg.MethodsA retrospective review of patients with renal biopsies in Guangdong Provincial People’s Hospital from 2005 to 2018 was performed. Clinicopathological data, treatments and remission of proteinuria were collected and compared between HBsAg+ and HBsAg- group.ResultsA total of 101 HBV-GN were recruited. Serum HBsAg+ and HBsAg- patients accounted for 62.4% and 37.6%, respectively. HBsAg+ group had poor kidney and liver functions. Pathological data showed the percentage of membranous nephropathy in HBsAg- group is significantly higher than that of HBsAg+ group (60.3% HBsAg+ vs 89.5% HBsAg-, P<0.05). Chronic renal tubular/interstitial injury was more prevalent in HBsAg+ group (16.9% HBsAg+ vs 2.6% HBsAg-, P<0.05). The deposition sites of immune complexes were significant different between the two groups. In addition, more HBsAg+ patients were given anti-HBV and less were given corticosteroid or immunosuppressants for treatment than that of HBsAg- patients. Percentages of clinical remission were increasing in both HBsAg+ and HBsAg- patients from 1, 3, 6 months to 1 year (18.75%, 45.2%, 67.8%, 82.4% vs 24.4%, 41.2%, 62.8%, 59.3%). The differences of remission betwen two groups were not significant (P>0.05).ConclusionThe clinicopathological characteristics and treatments of HBV-GN with serum HBsAg+ and HBsAg- were distinct, which indicated that the pathogenesis might be different and specific treatments were needed for HBV-GN patients with different serum HBsAg.

Role of histone demethylase KDM6B in HBx-mediated podocyte-macrophage transdifferentiation

Objective: To explore the expression of lysine (k)-specific demethylase 6B (KDM6B) in the renal tissues of hepatitis B virus-associated glomerulonephritis (HBV-GN) patients and human podocytes transfected with hepatitis B virus X (HBx) gene, and its role in HBx-mediated podocyte-macrophage transdifferentiation (PMT). Methods: Forty-eight patients diagnosed as HBV-GN by renal biopsy from 2013 to 2018 at the Shanghai Jiaotong University Affiliated First People's Hospital were included in this study. Thirty patients with primary glomerulonephritis (PGN) and fifteen patients with renal tumor were chosen as control group. The expression of KDM6B and macrophage marker F4/80 in renal tissues of HBV-GN patients was observed by immunofluorescence and immunohistochemistry. The association between kidney KDM6B levels and clinical features of HBV-GN patients was analyzed. The expression of KDM6B, F4/80, major histocompatibility complex (MHC)-Ⅱ and CD40 in the podocytes was detected by Western blotting. The contents of interferon-γ (IFN-γ) and interleukin-6 (IL-6) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The small interfering RNA of KDM6B (KDM6B siRNA) was used to silence the expression of KDM6B and the protein levels of KDM6B, F4/80 and tri-methylation of lysine 27 of histone H3 (H3K27me3) induced by HBx gene transfection were detected by Western blotting. Results: Renal KDM6B expression was significantly increased in HBV-GN patients compared to normal control (0.022±0.004 vs 0.006±0.002, P=0.006). There was no significant difference in the positive rate of KDM6B among different pathological types of HBV-GN (P=0.139). Moreover, co-expression of KDM6B and F4/80 could be observed in the podocytes of HBV-GN patients. Patients with estimated glomerular filtration (eGFR)<60 ml·min-1·(1.73 m2)-1or proteinuria ≥ 3.5 g/day had a significantly higher renal KDM6B expression compared to control groups (all P<0.05). In addition, the expression of KDM6B, F4/80, MHC-II and CD40 was significantly up-regulated in the podocytes transfected with HBx gene (all P<0.05). The content of IFN-γ and IL-6 in the supernatant was significantly increased (all P<0.05). After gene silencing of KDM6B, the expression of F4/80 induced by HBx in the podocytes was significantly down-regulated, while the level of H3K27me3 was significantly increased (both P<0.05). Conclusions: HBx could induce KDM6B expression in podocytes and initiate PMT, thereby involving in the dysfunction of immune microenviroment in the renal tissues of HBV-GN.

Efficacy and Safety of Long-Term Corticosteroid Monotherapy in 26 Cases of Nephrotic Syndrome with Biopsy-Proven Membranous Nephropathy Induced by Seronegative Hepatitis B Virus-Associated Glomerulonephritis

Background: Hepatitis B virus-associated glomerulonephritis (HBV-GN) can occur in patients with negative HBV serological antigens. Little is known about the treatment of seronegative HBV-GN (sn HBV-GN). The aim of this prospective study was to evaluate the efficacy and safety of corticosteroids in the treatment of sn HBV-GN. Methods: Twenty-six patients with nephrotic syndrome induced by seronegative HBV-associated membranous nephropathy were enrolled. The patients were given methylprednisolone (0.8 mg/kg/day) for 12–24 weeks, tapered by a 2-mg reduction every 1–3 months. Patients were followed up for 6–36 months. Complete remission (CR) was defined as proteinuria <0.3 g/24 h. Partial remission (PR) was defined as proteinuria of 0.3–3.5 g/24 h that was reduced ≥50% of the baseline level. Results: The effective remission (including CR and PR) rates of nephrotic syndrome were 23.1%, 61.5%, 73.1%, 76.2%, 90.5%, and 81.0%, respectively, after 1, 3, 6, 12, 24, and 36 months. Nineteen patients achieved effective remission after 11.68 ± 7.15 months. The level of serum albumin improved from 24.34 ± 6.71 g/L at baseline to 39.61 ± 7.45 g/L at the 36th month significantly. After treatment, the level of serum Cr was similar to the baseline. Only 2 patients relapsed. The primary adverse reaction was infection. None of the patients showed evidence of HBV replication. Conclusion: The long-term middle-dose corticosteroid therapy without antiviral drugs is effective and safe for membranous sn HBV-GN patients. For sn HBV-GN patients, the monitoring of HBV DNA and HBV markers in the serum is necessary during the corticosteroid monotherapy. Trial Registration: The Chinese Clinical Trial Registry (ChiCTR1900022518).

Features of lymphocyte differentiation in ANCA-associated and virus-associated glomerulonephritis in children

Glomerulonephritis is a heterogeneous group of immuno-inflammatory diseases. The purpose of the study: to analyze the subpopulation composition of lymphocytes in children with various types of glomerulonephritis. 50 children aged 1 to 16 years were examined: 1 group — 17 children aged 1 to 16 years with ANCA-associated glomerulonephritis; 2 group — 33 patients aged 2 to 16 years with virus-associated glomerulonephritis. Diagnostics of the subpopulation composition of lymphocytes was performed by flow cytometry using non-flushing technology using a three-color combination of monoclonal antibodies. It was found that the development of ANCA-associated glomerulonephritis is accompanied by signs of immunodeficiency against the background of a relative increase in the content of T-lymphocyte subpopulations. Differentiation of lymphocytes in children with virus-associated glomerulonephritis is ambiguous: the process goes either along the path of activation of antiviral protection, or along the path of development of insufficient antiviral activity of immunocytes. In children with virus-associated glomerulonephritis, a decrease in the level of B-lymphocytes was found, correlating with hypoimmunoglobulinemia A, which is an additional factor in reducing resistance on the mucous membranes. The study of lymphocyte differentiation in the development of glomerulonephritis is important for identifying the main immunological mechanisms involved in the pathogenesis of these diseases.

Severity of Intrarenal Arterial Lesions Can Predict the Clinical Prognosis of Hepatitis B Virus-Associated Glomerulonephritis: A Retrospective Study

Background: Intrarenal arterial lesions (IALs) have been studied in immunoglobulin A nephropathy and lupus nephritis, but this has not been reported in hepatitis B virus-associated glomerulonephritis (HBV-GN). This study aims to investigate the prevalence and the role of IALs in HBV-GN. Methods: IALs were examined in kidney biopsy specimens from 205 patients with HBV-GN retrospectively. The severity of IALs and tubular interstitial lesions was scored semi-quantitatively. The severity of IALs was divided into 4 groups on the basis of ILA score, which were no IALs (Score 0), mild IALs (Score 1–2), moderate IALs (Score 3–4), and severe IALs (Score 5–10) groups. Survival analysis was performed using the Kaplan-Meier method between the severity of IALs and clinical events (doubling of serum creatinine [SCr], ESRD, and death due to the kidney disease). Results: Among 205 patients with HBV-GN, 143 (69.8%) had IALs in their kidney biopsy specimens. IALs were mild in 28 (19.6%) patients, moderate in 101 (70.6%) patients, and severe in 14 (9.8%) patients. The severity of IALs was associated with high blood pressure (BP), high SCr, and severe tubulointerstitial injuries. The average follow-up time of these 205 HBV-GN patients was 94.2 ± 47.1 months, in which 46 cases had clinical event. The proportions of clinical events in no IAL, mild IAL, moderate IAL, and severe IAL groups were 9.7, 14.3, 25.7, and 71.4%, respectively. Event-free survival of patient in IAL group was significantly lower than that in the no IAL group (p = 0.000). Multivariate cox regression analysis indicated SCr (1.011, 1.007–1.016), hypertension (1.767, 1.004–3.108), and IAL (2.194, 1.062–4.530) were independent risk factors for clinical events after adjustment for age and gender. Event-free clinical survival in moderate and severe IAL groups was significantly lower than that in the no IAL group (p = 0.0111 and p = 0.0001, respectively). Besides, event-free renal survival in severe IAL group was significantly lower than that in moderate IAL group (p = 0.009). Multivariate cox regression analysis showed that the more severe the IALs, the higher the risk of the clinical event, with a hazard ratio of 2.284 for moderate IALs (1.085–4.907) and 3.315 for severe IALs (1.296–8.482). Conclusions: Severity of IALs is associated with high BP, reduced renal function, and poor clinical prognosis in HBV-GN patients.

Efficacy and safety of anti-viral therapy for Hepatitis B virus-associated glomerulonephritis: Ameta-analysis.

To assess the potency of anti-viral treatment for hepatitis B virus-associated glomerulonephritis (HBV-GN). Method: We searched for controlled clinical trials on anti-viral therapy for HBV-GN in MEDLINE, Embase, the Cochrane Library, and PubMed from inception to March 11th 2019. Seven trials, including 182 patients met the criteria for evaluating. The primary outcome measures were proteinuria and changes in the estimated glomerular filtration rate, and the secondary outcome measure was hepatitis B e-antigen clearance. A fixed or random effect model was established to analyze the data. Subgroup analyses were performed to explore the effects of clinical trial type, anti-viral drug type, age, and follow-up duration. The total remission rate of proteinuria (OR = 10.48, 95% CI: 4.60-23.89, I2 = 0%), complete remission rate of proteinuria (OR = 11.64, 95% CI: 5.17-26.21, I2 = 23%) and clearance rate of Hepatitis Be Antigen (HBeAg) were significantly higher in the anti-viral treatment group than in the control group (OR = 27.08, 95% CI: 3.71-197.88, I2 = 63%). However, antiviral therapy was not as effective regarding the eGFR (MD = 5.74, 95% CI: -4.24-15.73). In the subgroup analysis, age and drug type had significant impacts on proteinuria remission, and study type and follow-up duration only slightly affected the heterogeneity. Antiviral therapy induced remission of proteinuria and increased HBeAg clearance but failed to improve the eGFR. Pediatric patients were more sensitive to antiviral therapy than adults. IFNs seem more effective but are accompanied by more adverse reactions than NAs.

Efficacy of entecavir in treating hepatitis B virus-associated membranous nephropathy.

hepatitis B virus-associated membranous nephropathy (HBV-MN) is the most common pathological type of hepatitis B virus-associated glomerulonephritis. This study evaluated the efficacy of entecavir antiviral therapy for HBV-MN patients due to the intolerable side effects of interferon-alpha and high incidence rate of drug-resistance in lamivudine therapy. thirty-two patients with HBV-MN were identified by biopsy and treated with entecavir for 52 weeks. These patients were followed up to evaluate outcomes of entecavir-treatment. Descriptive statistics were used to summarize patient demographics and treatment outcomes. entecavir treatment reduced 24-h urinary protein excretion. The total probability of partial proteinuria and complete remission at 24 and 52 weeks was 53.1 and 78.1 %, respectively. A decrease of circulating HBV-DNA was observed in all patients with active HBV replication. The significant decrease of 24-h urinary protein began at 12 weeks, as early as the decrease of serum HBV-DNA level. The serum HBV DNA titers at baseline and after 52 weeks of treatment were 4.3 ± 2.8 log10 and 2.3 ± 1.7 log10, respectively. Meanwhile, eGFR increased from 100.3 ± 20.5 ml/min/1.73 m2 at baseline to 107.7 ± 15.9 ml/min/1.73 m2 after 52 weeks of treatment. The serum alanine aminotransferase level (ALT) gradually decreased to normal during entecavir antiviral treatment. entecavir treatment in HBV-MN patients was carefully described. Complete remission and HBV replication suppression were induced by entecavir treatment in HBV-MN patients. Patients with high serum creatinine (Scr), ALT and low eGFR levels benefit more from entecavir treatment. Entecavir therapy is well tolerated by patients and no adverse reactions were observed.

Telbivudine antagonizes TLR4 to inhibit the epithelial-to-mesenchymal transition in human proximal tubular epithelial cells in vitro

The antiviral drug Telbivudine (LdT) has an extrahepatic pharmaceutical effect that improves renal inflammation and tubulointerstitial fibrosis. However, the exact mechanism of action requires further investigation. Toll-like receptor 4 (TLR4) is involved in several physiological processes, including inflammation, fibrosis, innate immunity, and hepatitis B virus-associated glomerulonephritis. The epithelial-to-mesenchymal transition (EMT) is the characteristic pathological change in tubulointerstitial fibrosis. In this study, we used transforming growth factor-β (TGF-β) to stimulate human proximal tubular epithelial (HK-2) cells to investigate the effects of LdT in EMT. In addition, we treated HK-2 cells with a TLR4 agonist, lipopolysaccharide, to determine the effect of LdT on TLR4. The results indicated that LdT inhibited the expression of TLR4 and its downstream proteins. It also decreased the release of inflammatory factors, downregulated the TGF-β/Smad signaling pathway, and reversed the EMT changes seen in HK-2 cells. In conclusion, LdT antagonized TLR4 to inhibit EMT in proximal tubular epithelial cells.