Mesangial Cells Research Articles

MO242: Intrarenal Single Cell Sequencing of Hepatitis B Virus Associated Membranous Nephropathy

Abstract BACKGROUND AND AIMS The pathogenesis underlying hepatitis B virus (HBV)-associated membranous nephropathy (MN) remains incompletely understood. METHOD To decipher the etiopathogenesis of HBV-associated MN, intrarenal single-cell RNA sequencing (scRNA-seq) was applied to the kidney biopsy from a HBV-associated MN patient and a health control to define the transcriptomic landscape at single-cell resolution. Differentially expressed genes (DEGs) and enrichment analysis were compared between HBV-associated MN and anti-PLA2R positive idiopathic membranous nephropathy (IMN) using intrarenal scRNAseq. RESULTS We generated 4114 intrarenal single-cell transcriptomes from a HBV-associated MN patient by single-cell RNA-sequencing. We found that kidney podocytes cells in the HBV-associated MN patient displayed increased expression of several genes, including EDIL3, GPC6, HSPA5 and CTGF, which were closely related to extracellular matrix formation. Kidney endothelial cells of HBV-associated MN were mainly enriched in inflammatory pathways including TNF signaling, IL-17 signaling, NOD-like receptor signaling, NF-kappa B signaling pathway and Toll-like receptor signaling pathway. TNF signaling pathway and IL-17 signaling pathway activation were confirmed in proximal tubule cells from the HBV-associated MN patient. Moreover, ligand-receptor analysis highlighted the extensive communication of mesangial cells, which infers great importance in HBV-associated MN. Furthermore, gene ontology (GO) function enrichment analysis revealed that DEGs of endothelial cells from the HBV-associated MN patient were enriched in interleukin-6 secretion and interleukin-1 secretion when compared with anti-PLA2R positive IMN. CONCLUSION These results offer new insight into the pathogenesis of HBV-associated MN.

MO260: Prognostic Role of Electron Microscopy Lesions in IGA Nephropathy

Abstract BACKGROUND AND AIMS The progression of immunoglobulin A nephropathy (IgAN) is currently assessed using the Oxford MEST-C score, which uses five indicators (mesangial and endocapillary hypercellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy and the presence of crescents). However, little is known about the prognostic role of the glomerular lesions assessed by transmission electron microscopy (EM). METHOD We performed a unicentric retrospective study on 107 consecutive IgAN patients {age 42 [interquartile range (IQR) 33–54] years}, 67% male, eGFR 46 (IQR 33.3–65.2) mL/min, proteinuria 1.0 (IQR 0.4–2.1) g/g creatinine)—kidney biopsy-proven—between 2010 and 2015. Patients were followed until end-stage kidney disease (ESKD, dialysis or renal transplantation), death or end of study (January 2021), whichever came first. For each biopsy specimen, light microscopy, immunofluorescence and EM were routinely performed. One pathologist (GTB) reviewed independently the slides without knowledge of the original biopsy interpretation and assessed for MEST-C score and EM lesions according to the Mayo Clinic/Renal Pathology Society Consensus in podocytes, endothelial cells, mesangium and glomerular basement membrane (Table 1). RESULTS Patients were followed for a median of 7.1 (95% CI 6.7–7.5) years. The patients who reached ESKD—32 (43%)—had a higher Charlson comorbidity score [CCS 2 (IQR 2–4) versus 1 (IQR 0–3), P = .01)], arterial hypertension more often [88% versus 64%, P = .01)], lower eGFR [33.7 (IQR 22.4–44.3) versus 56.3 (IQR 39.4–72.5) mL/min, P < .001] and higher MEST-C score [2 (IQR 2–4) versus 1 (IQR 1–2), P < .001]. There were no differences regarding proteinuria, hematuria and treatment. In terms of EM lesions, patients who experienced ESKD had more frequent podocyte activation (47% versus 27%, P < 0.01), effacement (100% versus 83%, P = .01) and presence of microvilli (69% versus 35%, P = .001); more often endothelial cells activation (47% versus 27%, P = .04) and fenestration (56% versus 29%, P < 0.01); higher mesangial cells proliferation (91% versus 64%, P < .01). Mean kidney survival time for the entire cohort was 8.2 (95% CI 7.4–8.9) years. In univariate Cox proportional hazard (CPH) regression higher MEST-C score and EM lesions in podocytes, endothelial cells and mesangial cell proliferation were associated with a shorter kidney survival time (Table 1). However, in the multivariate CPH adjusted for CCS, arterial hypertension, eGFR and treatment, only higher MEST-C score, presence of podocytes with microvilli and mesangial cell proliferation were associated with ESKD (Table 1). CONCLUSION To the best of our knowledge, this is the first study to evaluate in depth the prognostic value of EM lesions in IgAN patients in a reasonable large cohort with an appropriate follow-up time. Besides the MEST-C score, we found that the presence of podocytes with microvilli and mesangial cell proliferation are associated with poor kidney survival.

FC074: Mineralocorticoid Receptor Drives Parietal Epithelial Cell Activation and Glomerular Injury During Crescentic Glomerulonephritis

Abstract BACKGROUND AND AIMS We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury (1). Accumulating evidence has indicated the potential contributions of aldosterone and mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is strongly expressed in endothelial cells, glomerular mesangial cells, podocytes and distal tubular cells. Previous studies have shown that administration of mineralocorticoid receptor antagonists, including spironolactone and eplerenone, has beneficial effects in various renal injury animal models, such as unilateral ureteral obstruction, ischemia-reperfusion, cyclosporine-induced nephrotoxicity and hypertensive renal injury. The role of the MR in extra capillary glomerulopathies is still elusive and mechanistically unclear. METHOD To investigate the cell-specific role of the MR in PEC in the course of crescentic glomerulonephritis, we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Crescentic glomerulonephritis (GN) was induced using the antiglomerular basement membrane nephrotoxic serum (NTS) model. In vivo, biological parameters (albuminuria, blood urea nitrogen—BUN), glomerular injury, as well as PEC activation (CD44, CD9 and fibronectin staining) were assessed. RESULTS At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged using the NTS model, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function as compared to Pec Cre Nr3c2 wt/wt counterparts. Crescents were also more numerous and organized in Pec Cre Nr3c2wt/wt mice. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less activation molecule CD44 on PECs. Thus, genetic disruption of MR in PEC as well as pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC and crescents. CONCLUSION Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis along with the recently discovered CD9/EGFR/PDGFR pathway. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

MO405: Leucine-Rich Alpha-2-Glycoprotein as a Potential Marker of Mesangial Cell Proliferation in Immunoglobulin a Nephropathy

Abstract BACKGROUND AND AIMS Leucine-rich alpha-2-glycoprotein (LRG1) is a novel proangiogenic factor involved in the abnormal angiogenesis and renal fibrosis in diabetic nephropathy by potentiating endothelial transforming growth factor-beta/activin receptor-like kinase 1 (TGF-β/ALK1) signalling [1]. TGF-β mediates mesangial matrix accumulation culminating in the development of glomerulosclerosis [2], which also occurs in immunoglobulin A nephropathy (IgAN) patients. Our study aims to evaluate LRG1 as a marker of mesangial cell proliferation in IgAN patients. METHODS: Adults with a morphologically confirmed IgAN by the presence of IgA deposits by immunofluorescence microscopy were included in the study. Diabetes mellitus, oncology, acute inflammation processes were exclusion criteria. Serum LRG1 was detected using ELISA. RESULTS A total of 73 patients with IgAN were included [mean age 41 years, interquartile range (IQR) 35–47; range, 21–65; 65.8% men]. The median estimated glomerular filtration rate (eGFR) was 44 (IQR 24–83, range 4–133) mL/min/1.73m2. Kidney biopsies were analyzed according to the Oxford classification: M0 in 10 (13,7%), M1 in 56 (76.7%) patients. The median serum LRG1 was 49 µg/mL (IQR 40.7–57.7, range 29–133.3). Bivariate analyses showed that plasma LRG1 was negatively associated with eGFR (rho = −0.342, P = 0.003). A linear model showed that GFR decline for 1 mL/min was associated with an increase of LRG1 for 0.194 µg/mL. In IgAN patients with an M1-score LRG1 was higher (P = 0027, Mann–Whitney U test), the area under the ROC curve value for serum LRG1 was 0.739. CONCLUSION Serum LRG1 increases with GFR decline in patients with IgAN and at the same time correlates with mesangial hypercellularity according to Oxford classification. Therefore, LRG1 might be a potential marker of advanced disease in IgAN.

MO174: Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposits: Rare Disease with Poor Prognosis?

Abstract BACKGROUND AND AIMS Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMIDs) is a recently described disease among the spectrum of monoclonal gammopathy of renal significance (MGRS). The disease is renal limited and manifests with chronic glomerular disease, altered renal function and albuminuria, sometimes in the nephrotic range. Kidney injury results from direct glomerular deposition of the monoclonal immunoglobulin. Of note, in approximately 70% of the cases of PGNMID, a clone cannot be detected. METHOD Clinical case of PGNMID. RESULTS A 36-year-old woman, with a past history of acute glomerulonephritis, was treated with mycophenolate mofetil 2 g and concomitant methylprednisolone 2 mg from January 2020 to September 2021 (kidney biopsy was not performed). After 3 months, she showed recurrence of nephrotic syndrome: peripheral edema, severe weakness, shortness of breath, decreased diuresis up to oliguria and increased blood pressure. Blood analysis has shown: GFR 127 mL/min/1.73 m2, total protein 44.4 g/L, albumin 16.2 g/L; autoimmune markers—negative; kappa free light chains 182 mg/L (3.3–19.4 mg/L); lambda free light chains 112 mg/L (5.71–26.3 mg/L), ratio of kappa/lambda 1.63 (0.26 to 1.65). Urine analysis has shown: total protein excretion rate 3.5 g/24 h; albuminuria 1000 mg/g; urine microscopy—normal; kappa free light chains ++++; lambda free light chains +++. A kidney biopsy has shown up to 12 glomerulies, which have different sizes, some are enlarged, as due to diffuse proliferation of mesangial cells, oedema and segmental proliferation of endothelial cells, as fibrinoid changes; some of the capillary loops are infiltrated with monocytes, an uneven increase in the mesangial matrix, there are small areas of segmental sclerosis that tend to be nodular in some glomerulies. Degenerative changes (granular, vacuolar, in some areas hyaline-droplet dystrophy), necrosis of epithelial cell groups, areas of epithelial atrophy with thickening of the tubular basement membrane (up to 10% of the biopsy volume) are sharply expressed in the tubules. In the tubules—protein masses, exfoliated epithelium, the lumen of the tubules is sharply reduced and/or absent due to pronounced degenerative changes in the epithelium. In the stroma—edema, areas of interstitial fibrosis, including perivascular (up to 10% of the biopsy volume), focal lymphohistiocytic cell infiltrates. By definition, there is evidence of monoclonal deposits by immunofluorescence: IgA (predominantly), IgG, IgM, С3 deposits; С1q, CD20 (В- lymphocytes), AA amyloidosis -negative expression; kappa free light chains (predominantly) +++; lambda free light chains ++ (Figure 1). Finally, we reached a clinical diagnosis of CKD G1, A3: PGNMIDs (IgM, IgA, IgG; LC Ķ˃λ). Nephrotic syndrome. Light Chain Deposition Disease. At that point (January 2021), we have started clone-targeted chemotherapy following the data sheet, with urinary and haematological response after 2 weeks, and normal kidney function with complete remission of nephrotic syndrome after 4 weeks, as well as a negativization of kappa and lambda free light chains deposits (Table 1). Three months after admission, the patient maintained haematological and kidney remission up to now. CONCLUSION MGRS is not an independent kidney disease, not ‘chronic glomerulonephritis’, but a condition in which kidney damage is secondary to clonal B-cell proliferation. In other words, MGRS is a precancerous disease combined with chronic kidney disease that requires immediate treatment. Renal prognosis is poor, with progression to ESKD in 25% of patients within 30 months and frequent early recurrence on the renal allograft. However, recent studies indicate that clone-targeted chemotherapy may significantly improve renal outcomes, opening future perspectives for the management of this rare disease.

MO609: Intrarenal TNF-α Expression in The Development of Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Diabetic nephropathy (DN) develops in 40%–60% of people with type 2 diabetes mellitus (DM) and is characterized by a progressive and persistent decline in renal function, being the main cause of end-stage renal disease worldwide. TNF-α plays an important role in the development of inflammation, can lead to the destruction of connective tissue, release of lytic enzymes, has a direct effect on the processes of tissue regeneration, changing the expression of collagen types I, III and V. Also, TNF-α stimulates an increase in the synthesis of endothelin-1 by glomerular mesangial cells and has a direct cytotoxic effect on renal tissue, and increased cytokine production induces the processes of apoptosis and necrosis of glomerular cells. The aim of this work was to study the role of intrarenal TNF-α expression in the development of morphological changes in renal tissue in DN patients. METHOD We examined 50 patients with type 2 diabetes (mean age 66.58 ± 3.27 years). The duration of DM is 17.69 ± 0.35 years, and the duration of DN from the moment of detection of albuminuria to morphological examination of the renal tissue and diagnosis is 1.63 ± 0.34 years. Light and immunofluorescence microscopy of biopsy specimens of renal tissue of patients was carried out. Morphological changes were assessed in accordance with the Pathologic Classification of Diabetic Nephropathy (2010). According to light microscopy, 12 patients had class IIa (mild mesangial expansion), 13 patients had class IIb (severe mesangial expansion), 19 patients had class III (Kimmelstiel–Wilson nodular lesions) and 5 patients had class IV (extended diabetic glomerulosclerosis). In all patients, TNF-α expression in the glomerulus and in the interstitium was determined by immunofluorescence microscopy. RESULTS Correlation analysis showed the effect of TNF-α expression in the glomerulus in class IIa on the development of segmental sclerosis of the glomeruli (P = 0.04). A decrease in TNF-α expression in the glomerulus is accompanied by periglomerular sclerosis (P = 0.012), and also increases: thickening of the glomerular basement membrane (P = 0.03), expansion of the mesangium passes from soft to severe and affects more than 25% of the mesangial matrix (IIb stage) (P = 0.033), Kimmelstiel–Wilson nodular lesions are formed (P = 0.009). The interstitial expression of TNF-α remains high in the epithelium of the urinary tubules and in the interstitial cells of the kidney, regardless of the DN class and influences the development of tubulointerstitial fibrosis (P = 0.003). In order to clarify the prognostic significance of TNF-α and its role in the progression of DN, a linear regression analysis was carried out with the calculation of the coefficients of determination R2 (R Square) and using the Durban–Watson test and analysis of variance (ANOVA "Analysis of Variance") using the F criterion and calculating the standardized beta coefficient (ß) with a 95% confidence interval. It has been shown that glomerular expression of TNF-α affects the following: (i) progression of the expansion of the mesangial matrix (F = 4.817, P = 0.033; β = –0.311; CI –0.506 to –0.22, P = 0.033); (ii) the formation of Kimmelstiel–Wilson nodular lesions (F = 5.383, P = 0.025; β = –0.327; CI –1.375 to –0.97, P = 0.025). Interstitial expression of TNF-α contributes to the following: (i) the progression of arterial hyalinosis (F = 4.349, P = 0.043; β = 0.297; CI 0.009–0.497, P = 0.043); (ii) interstitial sclerosis (F = 9.541, P = 0.003; β = 0.422; CI 0.101–0.479, P = 0.003). CONCLUSION

Salvianolic acid B attenuates membranous nephropathy by activating renal autophagy via microRNA-145-5p/phosphatidylinositol 3-kinase/AKT pathway

ABSTRACT The abnormal proliferation and inflammatory response of the mesangial cells play a crucial role in the progression of membranous nephropathy (MN). Herein, this study aimed to investigate the therapeutic effect of Salvianolic acid B (SalB) on MN-induced mesangial abnormalities and its underlying mechanisms. MN models were established in cationic bovine serum albumin-induced Sprague-Dawley rats and lipopolysaccharide-induced human mesangial cells (HMCs). Following SalB and microRNA-145-5p antagomir treatment, kidney function was investigated by 24-hours urine protein, serum creatinine, and blood urea nitrogen. Pathological changes of kidney were investigated by Periodic acid Schiff staining. CD68 and IgG were detected by immunofluorescence in glomerulus. Mesangial autophagosomes were observed by transmission electron microscope. MicroRNA-145-5p inhibitor, mimic, LY294002, and SalB were used to treat with HMCs. In kidney and HMCs, IL-1 , IL-2, IL-6, TNF- and microRNA-145-5p was detected by quantitative real-time PCR. Phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, AKT, beclin1, and microtubule-associated protein light chain 3 (LC3) levels were detected by Western blot. HMCs proliferation and cycle were detected by Cell Counting Kit-8 and flow cytometry. LC3 were detected by LC3-dual-fluorescent adenovirus in HMCs. Our results showed that SalB significantly ameliorated kidney function and pathological changes. Furthermore, it significantly alleviated proliferation, inflammation and activated autophagy in mesangial cells. Moreover, microRNA-145-5p antagomir accentuated MN while microRNA-145-5p inhibitor and LY294002 encouraged proliferation and inflammation through PI3K/AKT pathway in HMCs. Collectively, our study demonstrated that SalB activated renal autophagy to reduce cell proliferation and inflammation of MN, which was mediated by microRNA-145-5p to inhibit PI3K/AKT pathway, and ultimately attenuated MN.

JNK and Jag1/Notch2 co-regulate CXCL16 to facilitate cypermethrin-induced kidney damage

Cypermethrin (CYP), a widely-used composite pyrethroid pesticide, has underlying nephrotoxic effects. To elucidate potential roles of the MAPK pathway, the Jag/Notch pathway, and miRNAs in CYP-mediated kidney lesion, Sprague-Dawley rats and glomerular mesangial cells were used in this work. Results displayed that β-CYP abnormally altered renal histomorphology and ultrastructures, induced renal DNA damage, and impaired renal functions, as evidenced by the increase in plasma levels of Cys-C and β2-Mg. β-CYP activated the JNK/c-Jun pathway by inducing ROS and oxidative stress. Meanwhile, β-CYP changed the miRNA expression profile, miR-21–5p showing the most significant increase. Moreover, the Jag1/Notch2/Hes1 pathway was directly targeted by miR-21–5p, the mRNA and protein expression of Jag1, Notch2, and Hes1 being declined in vivo and in vitro. The chemokine CXCL16 was induced by β-CYP, accompanied by the inflammatory factor production and inflammatory cell infiltration in kidneys. The specific JNK inhibitor, Jag1 overexpression, Hes1 overexpression, bidirectional Co-IP, ChIP, and CXCL16 silencing demonstrated that CXCL16 co-regulated by the JNK/c-Jun and Jag1/Notch2/Hes1 pathways elicited renal inflammation. Collectively, our findings indicate that β-CYP is of nephrotoxicity and it not only directly changes renal histomorphology and ultrastructures, but induces CXCL16 to trigger renal inflammation via the JNK/c-Jun and Jag1/Notch2/Hes1 pathways, finally synergistically contributing to kidney damage.

Calcium‐Dependent Dual Oxidase 2 is a Novel Source of Reactive Oxygen Species Implicated in Glomerular Mesangial Cell Fibrotic Response to Angiotensin II and Glucose

The vasoactive peptide angiotensin II (Ang II) and hyperglycemia contribute to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II or high concentrations of glucose (HG). Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression, along with its associated maturation factor, DuoxA2, are upregulated by Ang II and HG.Small interfering RNA (siRNA)‐mediated downregulation of Duox2 significantly reduces Ang II‐induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a‐smooth muscle actin and fibronectin expression).To demonstrate that Duox2 activation is Ca2+‐dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II‐induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura‐2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II‐stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury.In summary, we have identified a novel role for Duox2 and its cognate maturation factor, DuoxA2, as a major source of ROS in response to Ang II and HG and established the significance of Duox2 in Ang II‐mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.

The role of autophagy in cadmium-induced acute toxicity in glomerular mesangial cells and tracking polyubiquitination of cytoplasmic p53 as a biomarker

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.

Circulating extracellular vesicles from patients with severe COVID-19 upregulate Cathepsin B and activate STAT3 in normal human mesangial cells

Abstract Severe COVID-19 disease is associated with acute kidney injury in up to 57% of infected patients. The etiology of this is likely multifactorial. Extracellular vesicles (EVs) contain unique molecular cargo that contributes to health and disease in many different organ systems and can contain useful surrogate biomarkers. To gain insight into SARS-CoV2’s impact on the human kidney, we prospectively collected blood and urine from individuals hospitalized with severe COVID-19 infection and isolated EVs. Using these EVs, we investigated the impact of circulating EVs released during COVID-19 infection on healthy human glomerular mesangial cells. Nanoparticle tracking analysis and electron microscopy showed that EVs isolated from the plasma of COVID-19 patients were larger in size compared to those isolated from the plasma of non-COVID-19 patients, suggesting the presence of unique cargo within circulating EVs in the setting of COVID-19. The plasma derived EV’s from COVID-19 patients also increased glomerular cell proliferation, STAT3 activation, and cathepsin B protein expression in normal human glomerular cells when compared to EVs isolated from the plasma of non-COVID-19 patients. These data suggest systemic EVs during COVID-19 infection alter normal human mesangial cell function. and provide insight into the effects of SARS-CoV2 on the kidney. This work was supported by the University of Florida College of Medicine and the University of Mississippi Medical Center.

The Polysaccharide of Ligustrum Lucidum Ait. Protected from Renal Fibrosis by Interfering with Angiotensin II Signaling

BackgroundA number of preclinical advances have indicated the cellular and molecular mechanisms underlying renal fibrosis, such as the signaling of angiotensin II (Ang II), which is a bioactive peptide in renin‐angiotensin system. As one of kidney‐tonifying herbs, Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait. (Oleaceae) recorded as a tonic and dietary supplement, is a commonly prescribed component in traditional kidney‐tonifying herbal formula. This study aimed to clarify the polysaccharide component in FLL and investigate its potential role in preservation against renal fibrosis.MethodsThe crude polysaccharide of FLL was prepared and deproteinized by Sevag reagent. After lyophilization, the DEAE‐cellulose DE52 column and the Sephacryl S‐200 HR column were used for further isolation and purification of homogenous polysaccharide. The mice with unilateral ureteral obstruction (UUO) underwent daily intraperitoneal injection with FLL polysaccharide starting 7 days before UUO surgery until day 7 after surgery. The murine mesangial SV40‐MES13 cells were treated with vehicle, Ang II (10‐7 M) either with the absence and presence of angiotensin type I receptor blocker (ARB) (10‐6M), or with low dose (50 μg/mL, Poly‐L) and high dose (200 μg/mL, Poly‐H) of FLL polysaccharide.ResultsThe purified homogenous polysaccharide (Poly) was explored from FLL and the carbohydrate content was 97.45%. A hypothetical structure for Poly was proposed considering the comprehensive data of methylation, monosaccharides, and NMR analyses. Treatment of UUO mice with Poly decreased kidney index and ameliorated glomerular sclerosis and interstitial fibrosis, as well as down‐regulated the expression of profibrogenic factor VEGF and matrix fibrotic protein fibronectin (FN). Furthermore, as the same effect to ARB, the pre‐incubation with Poly alleviated the Ang II‐induced up‐regulation of FN in mesangial cells by immunofluoresence staining. The protein expressions of FN and proinflammatory factors like MCP‐1 and Rantes were significantly lower in Poly‐treated cell group than those in Ang II treatment group.ConclusionThe polysaccharide of Ligustrum lucidum Ait. was accurately explored with structure clarification. This polysaccharide component repressed angiotensin II‐induced fibrotic phenotype of renal mesangial cells and exerted beneficial effects on kidney of UUO mice.

Vitamin D Regulates Natriuretic Peptide Receptor‐A Gene Expression via Modulation of Histone Deacetylases and DNA Methyltransferase Activity

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces the second messenger cGMP. cGMP activates downstream signaling and biological consequences of ANP/NPRA such as natriuresis, diuresis, vasorelaxation, antimitogenic responses, and cardiac and renal anti‐hypertrophic effects. The activity and expression of Npr1 gene (coding for GC‐A/NPRA) assessed primarily through ANP‐stimulated cGMP production, are mediated by agents, including autoregulation involving natriuretic peptides, osmotic sensors, and other external and internal stimuli, but the hormonal and epigenetic mechanisms mediating Npr1 regulation are not well understood. The objective of present study was to study the effect of Vitamin D (vitD) in the regulation of Npr1 gene transcription and expression via modulation of epigenetic factors. Our bioinformatics study with the murine Npr1promoter has shown presence of 4 vitD response elements (VDRE) in the region ‐583 to ‐495 from the transcription start site having perfect and VDRE‐like consensus sequence. Npr1promoter activity was determined by transient transfection of Npr1 promoter deletion constructs in cultured rat thoracic aortic smooth muscle cells (RTASMCs) and mouse mesangial cells (MMCs) treated with Vitamin D₃ (1α, 25‐dihydroxy; VD3) using dual luciferase assay kit. Luciferase assay demonstrated that VD3 enhances Npr1 promoter activity by more than 5‐6‐fold in RTASMCs and MMCs in a dose‐dependent manner. Western blot and densitometry analysis showed increasing concentrations of VD3 significantly induced NPRA protein levels by 3.5‐fold in MMCs and 4.5‐fold in RTASMCs and maximum effect was observed at 100 nM. There was 45%‐50% inhibition of histone deacetylase (HDAC) activity in presence of VD3 in both cell lines as measured by HDAC activity/inhibition ELISA kit. Moreover, treatment with VD3 reduced class I HDAC enzymes, HDAC1 and HDAC3 protein levels and dose‐dependently enhanced acetylation level of histones, H3 at lysine residues 9 and 14 (H3‐K9/14 ac) and H4 at lysine residue 12 (H4‐K14ac). Treatment of RTASMCs and MMCs with VD3 attenuated DNA methyltransferase (DNMT) activity by 40%‐55% as measured by DNMT activity/inhibition ELISA kit. The results demonstrate that VD3 regulate Npr1 gene expression by modulation of histone deacetylases and DNMT activity. The identification of epigenetic targets of vitamin D signaling as a regulator of Npr1gene transcription and protein expression will have important implications in hypertension and cardiovascular regulation.

Comparison of clinical and pathological features of secondary IgA nephropathy of ankylosing spondylitis and primary IgA nephropathy.

Comparison of clinical and pathological features of secondary IgA nephropathy of ankylosing spondylitis (SIgAN-AS) and primary IgA nephropathy (PIgAN). Clinical characteristics and pathological data of patients diagnosed with IgAN by renal biopsy were collected in our hospital from January 2008 to October 2018. Patients with SIgAN-AS and PIgAN were recruited at a ratio of 1:5. Fifteen patients with SIgAN-AS and 75 patients with PIgAN were enrolled in this retrospective study. There were 15 cases in the SIgAN-AS group, including 13 males and 2 females. The cohort of 75 patients with PIgAN included 37 males and 38 females. There were significantly more males in the SIgAN-AS group 13/15 (86.67%) vs 37/75 (49.30%), p < 0.05. Compared with PIgAN patients, SIgAN-AS patients had higher incidences of hematuria (12/15 (80.00%) vs. 37/75 (49.33%), p<0.05), lower levels of 24-hour urinary protein (0.85±0.68 vs. 1.57±1.54g, p<0.05), but higher levels of estimated glomerular filtration rate (eGFR) (CKD-EPI formula; 112.56±24.53 vs. 88.23±29.39, p<0.05), albumin (44.67±3.48 vs. 41.09±7.07 g/L, p<0.05), erythrocyte sedimentation rate (ESR) (43.20±33.94 vs. 18.79±16.26mm/h, p<0.001), and C-reactive protein (CRP) (21.19±30.61 vs. 2.11±4.58mg/L, p<0.001). From the perspective of renal pathology of PIgAN, patients with SIgAN-AS had a lower incidence of renal tubular atrophy/interstitial fibrosis of nephropathy (p<0.05). Immunohistostaining analysis showed a higher incidence of dominant deposits of single IgA in the mesangial cell area (p<0.05). SIgAN-AS was more commonly observed in males and displayed a milder progression than those PIgAN. The majority of SIgAN-AS patients can be improved by early intervention.

Effect of Combined Mycophenolate and Rapamycin Treatment on Kidney Fibrosis in Murine Lupus Nephritis.

Background: A significant proportion of lupus nephritis patients develop chronic kidney disease (CKD) and progressive kidney fibrosis, for which there is no specific treatment. We previously reported that mycophenolate or rapamycin monotherapy showed comparable efficacy in suppressing kidney fibrosis in a murine model of lupus nephritis through their direct action on mesangial cells. We extended our study to investigate the effect of combined mycophenolate and rapamycin treatment (MR) on kidney fibrosis in NZBWF1/J mice. Methods: Female NZBWF1/J mice with active nephritis were randomized to receive vehicle or treatment with mycophenolate (50 mg/kg/day) and rapamycin (1.5 mg/kg/day) (MR) for up to 12 weeks, and the effect of treatment on clinical parameters, kidney histology, and fibrotic processes was investigated. Results: Progression of nephritis in untreated mice was accompanied by mesangial proliferation, glomerulosclerosis, tubular atrophy, protein cast formation, increased mTOR and ERK phosphorylation, and induction of TGF-β1, IL-6, α-smooth muscle actin, fibronectin, and collagen expression. Combined MR treatment prolonged survival, improved kidney function, decreased anti-dsDNA antibody level, and ameliorated histopathological changes. The effect of combined MR treatment on kidney histology and function was comparable to that of mycophenolate or rapamycin monotherapy. In vitro studies in human mesangial cells showed that exogenous TGF-β1 and IL-6 both induced mTOR and ERK phosphorylation and downstream fibrotic processes. Both mycophenolic acid and rapamycin inhibited inflammatory and fibrotic processes induced by TGF-β1 or IL-6 by downregulating mTOR and ERK phosphorylation. Conclusions: Our findings indicate that combined mycophenolate and rapamycin, at reduced dose, improves kidney fibrosis in murine lupus nephritis through their distinct effect on mTOR and ERK signaling in mesangial cells.

Long Non-Coding NONRATG001910.2 Promotes the Proliferation of Rat Mesangial Cell Line HBZY-1 Through the miR-339-3p/CTNNB1 Axis.

Chronic glomerulonephritis (CGN) is one of the leading causes of end-stage renal disease (ESRD). A growing body of literature emphasizes the important role of long non-coding RNAs (lncRNAs) in the development and progression of the disease. However, the function of NONRATG001910.2 in the development of CGN was not well understood. This research aimed to investigate the effect of NONRATG001910.2 on CGN and revealed its potential molecular mechanisms. In this work, the expression of NONRATG001910.2 was detected by quantitative real-time polymerase chain reaction (qRT-RCR) in cell lines. We found that NONRATG001910.2 was significantly up-regulated in lipopolysaccharide (LPS) induced cells. High NONRATG001910.2 levels were associated with the development of CGN. In addition, NONRATG001910.2 knockdown inhibited cell proliferation and cell cycle. At the same time, we found that up-regulation of microRNA-339-3p (miR-339-3p) abrogated the biological roles of NONRATG001910.2 up-regulation. Moreover, the knockdown of CTNNB1 can upregulate miR-339-3p expression, thereby inhibiting cell proliferation. In conclusion, these results demonstrated that NONRATG001910.2 in LPS-stimulated rat mesangial cell line HBZY-1 (HBZY-1) by targeting miR-339-3p, which subsequently promotes the expression of CTNNB1, and suggested that NONRATG001910.2 may be a potential biomarker.

The potential role of complement alternative pathway activation in hypertensive renal damage.

Hypertensive renal damage is a common secondary kidney disease caused by poor control of blood pressure. Recent evidence has revealed abnormal activation of the complement alternative pathway (AP) in hypertensive patients and animal models and that this phenomenon is related to hypertensive renal damage. Conditions in the setting of hypertension, including high renin concentration, reduced binding of factor H to the glomerular basement membrane, and abnormal local synthesis of complement proteins, potentially promote the AP activation in the kidney. The products of the AP activation promote the phenotypic transition of mesangial cells and tubular cells, attack endothelial cells and recruit immunocytes to worsen hypertensive renal damage. The effects of complement inhibition on hypertensive renal damage are contradictory. Although clinical data support the use of C5 monoclonal antibody in malignant hypertension, pharmacological inhibition in hypertensive animals provides little benefit to kidney function. Therefore, the role of the complement AP in the pathogenesis of hypertensive renal damage and the value of complement inhibition in hypertensive renal damage treatment must be further explored.

Anti-Inflammatory Effect of Resveratrol Derivatives via the Downregulation of Oxidative-Stress-Dependent and c-Src Transactivation EGFR Pathways on Rat Mesangial Cells.

In Taiwan, the root extract of Vitis thunbergii Sieb. et Zucc. (Vitaceae, VT) is rich in stilbenes, with resveratrol (Res) and its derivatives being the most abundant. Previously, we showed that the effect of Res derivatives against tumor necrosis factor-α (TNF-α)-stimulated inflammatory responses occurs via cPLA2/COX-2/PGE2 inhibition. This study compared and explored the underlying anti-inflammatory pharmacological mechanisms. Before stimulation with TNF-α, RMCs were treated with/without pharmacological inhibitors of specific protein kinases. The expression of inflammatory mediators was determined by Western blotting, gelatin zymography, real-time PCR, and luciferase assay. Cellular and mitochondrial ROS were measured by H2DHFDA or DHE and MitoSOX™ Red staining, respectively. The RNS level was indirectly measured by Griess reagent assay. Kinase activation and association were assayed by immunoprecipitation followed by Western blotting. TNF-α binding to TNFR recruited Rac1 and p47phox, thus activating the NAPDH oxidase-dependent MAPK and NF-κB pathways. The TNF-α-induced NF-κB activation via c-Src-driven ROS was independent from the EGFR signaling pathway. The anti-inflammatory effects of Res derivatives occurred via the inhibition of ROS derived from mitochondria and NADPH oxidase; RNS derived from iNOS; and the activation of the ERK1/2, JNK1/2, and NF-κB pathways. Overall, this study provides an understanding of the various activities of Res derivatives and their pharmacological mechanisms. In the future, the application of the active molecules of VT to health foods and medicine in Taiwan may increase.

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.

Aqueous Extract and Polysaccharide of Aconiti Lateralis Radix Induce Apoptosis and G0/G1 Phase Cell Cycle Arrest by PI3K/AKT/mTOR Signaling Pathway in Mesangial Cells.

Mesangial proliferative glomerulonephritis (MesPGN) is a common renal disease that lacks effective drug intervention. Aconiti Lateralis Radix (Fuzi), a natural Chinese medical herb, is found with significant therapeutic effects on various diseases in the clinic. However, its effects on MesPGN have not been reported. This study is aimed to discuss the therapeutic effects of the aqueous extract of Aconiti Lateralis Radix (ALR) and the polysaccharides of Aconiti Lateralis Radix (PALR) on MesPGN as well as the underlying mechanism. In this study, we, firstly, studied the anti-MesPGN mechanism of ALR and PALR. ALR and PALR inhibit the proliferation of the mesangial cells through the PI3K/AKT/mTOR pathway, induce the G0/G1 phase of block and apoptosis, inhibit the activity of Cyclin E and CDK2, increase the expression of Bax, cleaved caspase-8/caspase-8, and cleaved caspase-3/caspase-3 proteins, and effectively inhibit the growth of the mesangial cells. Overall, our data suggest that ALR and PALR may be potential candidates for MesPGN and that PALR is more effective than ALR.