Activation of the IL-6/STAT3 pathway contributes to the pathogenesis of membranous nephropathy and is a target for Mahuang Fuzi and Shenzhuo Decoction (MFSD) to repair podocyte damage.

BACKGROUND: Primary membranous nephropathy (PMN) is an autoimmune glomerular disease. IL-6 is a potential therapeutic target for PMN. Previous clinical studies have demonstrated the effectiveness of Mahuang Fuzi and Shenzhuo Decoction (MFSD) in treating membranous nephropathy. However, the mechanism of action of MFSD remains unclear. METHODS: Serum IL-6 levels were measured in patients with PMN and healthy subjects. The passive Heymann nephritis (PHN) rat model was established, and high and low doses of MFSD were used for intervention to observe the repair effect of MFSD on renal pathological changes and podocyte injury. RNA-seq was used to screen the possible targets of MFSD, and the effect of MFSD targeting IL-6/STAT3 was further verified by combining the experimental results. Finally, the efficacy of tocilizumab in PHN rats was observed. RESULTS: Serum IL-6 levels were significantly higher in PMN patients than in healthy subjects. These levels significantly decreased in patients in remission after MFSD treatment. MFSD treatment improved laboratory indicators in PHN rats, as well as glomerular filtration barrier damage and podocyte marker protein expression. Renal transcriptome changes showed that MFSD-targeted differential genes were enriched in JAK/STAT and cytokine-related pathways. MFSD inhibits the IL6/STAT3 pathway in podocytes. Additionally, MFSD significantly reduced serum levels of IL-6 and other cytokines in PHN rats. However, treatment of PHN with tocilizumab did not achieve the expected effect. CONCLUSION: The IL-6/STAT3 signaling pathway is activated in podocytes of experimental membranous nephropathy. MFSD alleviates podocyte damage by inhibiting the IL-6/STAT3 pathway.

Celastrol ameliorates lupus by promoting apoptosis of autoimmune T cells and preventing autoimmune response in MRL/lpr mice.

OBJECTIVE: Celastrol is a bioactive constituent extracted from Tripterygium wilfordii (thunder god vine). It has been demonstrated to have a therapeutic effect on experimental disease models for chronic inflammatory and immune disorders. In the present study, we investigated whether and how celastrol exerts a regulatory effect on the autoimmune response in MRL/lpr mice. METHODS: We performed an in vivo study to determine the therapeutic effects of celastrol in MRL/lpr mice and then further investigated the underlying mechanism of celastrol in the regulation of the autoimmune response in MRL/lpr mice. RESULTS: Celastrol showed a therapeutic effect in MRL/lpr mice by preventing the enlargement of the spleen and lymph nodes, alleviating renal injury, and reducing the levels of ANA and anti-double-stranded DNA antibodies. Furthermore, celastrol suppressed the in vivo inflammatory response in MRL/lpr mice by reducing the serum levels of multiple cytokines, including interleukin (IL)-6, tumour necrosis factor (TNF) and interferon (IFN)-γ, and the production of multiple antibody subsets, including total IgG, IgG1 and IgG2b. In vitro, celastrol reduced anti-CD3 antibody stimulation-induced T helper 1 and TNF-producing cells in CD4+ T cells of MRL/lpr mice. In addition, celastrol significantly affected B cell differentiation and prevented the generation of plasma cells from B cells in MRL/lpr mice by reducing the frequency of activated and germinal centre B cells. Celastrol treatment also affected T cell differentiation and significantly reduced central memory T cell frequencies in MRL/lpr mice. Importantly, celastrol treatment specifically promoted apoptosis of CD138+ but not CD138- T cells to suppress autoimmune T cell accumulation in MRL/lpr mice. CONCLUSIONS: Celastrol exerted therapeutic effects on lupus by specifically promoting apoptosis of autoimmune T cells and preventing the progression of autoimmune response.

Single cell landscape of parietal epithelial cells in healthy and diseased states.

The biology and diversity of glomerular parietal epithelial cells (PECs) are important for understanding podocyte regeneration and crescent formation. Although protein markers have revealed the morphological heterogeneity of PECs, the molecular characteristics of PEC subpopulations remain largely unknown. Here, we performed a comprehensive analysis of PECs using single-cell RNA sequencing (scRNA-seq) data. Our analysis identified five distinct PEC subpopulations: PEC-A1, PEC-A2, PEC-A3, PEC-A4 and PEC-B. Among these subpopulations, PEC- A1 and PEC-A2 were characterized as podocyte progenitors while PEC-A4 represented tubular progenitors. Further dynamic signaling network analysis indicated that activation of PEC-A4 and the proliferation of PEC-A3 played pivotal roles in crescent formation. Analyses suggested that upstream signals released by podocytes, immune cells, endothelial cells and mesangial cells serve as pathogenic signals and may be promising intervention targets in crescentic glomerulonephritis. Pharmacological blockade of two such pathogenic signaling targets, proteins Mif and Csf1r, reduced hyperplasia of the PECs and crescent formation in anti-glomerular basement membrane glomerulonephritis murine models. Thus, our study demonstrates that scRNA-seq-based analysis provided valuable insights into the pathology and therapeutic strategies for crescentic glomerulonephritis.

Level of interleukin-35 in patients with idiopathic membranous nephropathy and its predictive value for remission time.

Objective: As a member of interleukin-12 family, interleukin-35 (IL-35) plays an important regulatory role in immune response. The relationship between IL-35 and idiopathic membranous nephropathy (IMN) is still unclear, and the purpose of this study is to clarify the relationship between IL-35 and disease activity and remission of IMN. Methods: This study was a single-center, retrospective study in which all patients were diagnosed with IMN by renal biopsy or aPLA2R titer and treated with Mahuang Fuzi and Shenzhuo Decoction (MFSD). A follow-up was conducted with the endpoint of clinical complete or partial remission (CR+PR). Levels of serum IL-35 were measured and its relationship with IMN remission were analyzed. The regulatory T cell (Treg) and inducible IL-35 producing Tregs (iTR35) in peripheral blood of IMN patients were detected by flow cytometry. Results: A total of 76 IMN patients (age 51.95 ± 13.29) were followed-up for 18 (12, 24) months. The level of serum IL-35 in all patients increased after treatment, but the degree of increase in remission group was significantly higher than that in no remission (NR) group (117.6% vs 83.7%, P<0.01). The baseline IL-35 level in remission group was higher than that in NR group (174.87 vs.151.87 pg/ml, P=0.016). Cox regression analysis showed that baseline IL-35 level was a independent risk factor for IMN remission (HR 1.081, 95%CI 1.048-1.116, P<0.001). Patients with baseline IL-35 lower than the lower quartile (≤145.49 pg/ml) had an average remission time twice as long as those with baseline IL-35 higher than the upper quartile (> 203.05 pg/ml) (12mon vs. 24mon, P<0.01). The baseline IL-35 can predict the remission time of IMN patients with either aPLA2R positive (AUC=0.673) or negative (AUC=0.745). Analysis of 18 patients with IMN showed that IL-35 level had a higher correlation with iTR35, but not Treg (r=0.613, P<0.05). Conclusions: The level of IL-35 in patients with IMN showed an increasing trend with the progress of treatment, and the baseline IL-35 could predict the remission time of IMN patients, including those patients with negative aPLA2R. The level of IL-35 is related to the number of iTR35 cells.

Coronary artery bypass grafting vs. percutaneous coronary intervention in coronary artery disease patients with advanced chronic kidney disease: A Chinese single-center study.

Objectives: Aims to compare the contemporary and long-term outcomes of coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) in coronary artery disease (CAD) patients with advanced chronic kidney disease (CKD). Methods: 823 CAD patients with advanced CKD (eGFR < 30 ml/min/1.73 m2) were collected, including 247 patients who underwent CABG and 576 patients received PCI from January 2014 to February 2021. The primary endpoint was all-cause death. The secondary endpoints included major adverse cardiac and cerebrovascular events (MACCEs), myocardial infarction (MI), stroke and revascularization. Results: Multivariable Cox regression models were used and propensity score matching (PSM) was also performed. After PSM, the 30-day mortality rate in the CABG group was higher than that in the PCI group but without statistically significant (6.6% vs. 2.4%, p = 0.24). During the first year, patients referred for CABG had a hazard ratio (HR) of 1.42 [95% confidence interval (CI), 0.41-3.01] for mortality compared with PCI. At the end of the 5-year follow-up, CABG group had a HR of 0.58 (95%CI, 0.38-0.86) for repeat revascularization, a HR of 0.77 (95%CI, 0.52-1.14) for survival rate and a HR of 0.88(95%CI, 0.56-1.18) for MACCEs as compared to PCI. Conclusions: Among patients with CAD and advanced CKD who underwent CABG or PCI, the all-cause mortality and MACCEs were comparable between the two groups in 30 days, 1-year and 5 years. However, CABG was only associated with a significantly lower risk for repeat revascularization compared with PCI at 5 years follow-up.

Cryoglobulinemic vasculitis and glomerulonephritis: concerns in clinical practice.

OBJECTIVE: Cryoglobulinemia often causes systemic vasculitis, thereby damaging to skin and internal organs including kidneys, even life-threatening. This review aimed to introduce the advances in understanding, detection, and treatment of this disease in recent years, with a particular concern to clinical practice. DATA SOURCES: All the data in this review were from the English or Chinese literature in the PubMed and China National Knowledge Infrastructure databases as of March 2019. STUDY SELECTION: This review selected important original articles, meaningful reviews, and some reports on cryoglobulinemia published in recent years and in history, as well as the guidelines for treatment of underlying diseases which lead to cryoglobulinemia. RESULTS: Diagnosis of cryoglobulinemia relies on serum cryoglobulin test, in which to ensure that the blood sample temperature is not less than 37°C in the entire pre-analysis phase is the key to avoid false negative results. Cryoglobulinemic vasculitis (Cryo Vas), including cryoglobulinemic glomerulonephritis (Cryo GN), usually occurs in types II and III mixed cryoglobulinemia, and can also be seen in type I cryoglobulinemia caused by monoclonal IgG3 or IgG1. Skin purpura, positive serum rheumatoid factor, and decreased serum levels of C4 and C3 are important clues for prompting types II and III Cryo Vas. Renal biopsy is an important means for diagnosis of Cryo GN, while membranous proliferative GN is the most common pathological type of Cryo GN. In recent years, great advances have been made in the treatment of Cryo Vas and its underlying diseases, and this review has briefly introduced these advances. CONCLUSIONS: Laboratory examinations of serum cryoglobulins urgently need standardization. The recent advances in the diagnosis and treatment of Cryo Vas and GN need to be popularized among the clinicians in related disciplines.

Inhibition of local aldosterone by eplerenone reduces renal structural damage in a novel model of chronic cyclosporine A nephrotoxicity.

BACKGROUND AND OBJECTIVE: The fact that mineralocorticoid receptor antagonists reduce structural and functional alterations induced by cyclosporine A (CsA) indicates that aldosterone plays a key role in chronic CsA nephrotoxicity. We and other researchers have reported local renal aldosterone synthesis. To investigate local renal aldosterone's role in chronic CsA nephrotoxicity, we evaluated the effect of eplerenone (Epl) on renal structural damage and renal dysfunction in adrenalectomized (ADX) rats, and assessed whether the therapeutic benefit was associated with reduction of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), plasminogen activator inhibitor type 1 (PAI-1) and collagen I (COL-I) expression. METHODS: Male Sprague-Dawley rats fed a normal-sodium diet were divided in four groups: sham-ADX, ADX, CsA, or Epl. Rats in the ADX, CsA and Epl groups were adrenalectomized first. Aldosterone, sodium and potassium levels in serum and urine were measured on the second day. Two weeks later, vehicle (sham-ADX and ADX group), CsA (25mg/kg/d), or CsA and Epl (100 mg/ kg/d) combination was administrated, respectively. After six weeks, urinary protein, creatinine clearance (Ccr), tubulointerstitial fibrosis (TIF), aldosterone level in kidney, and renal aldosterone synthase CYP11B2, COL-I, TGF-ß1, CTGF and PAI-1 gene expression levels were determined. RESULTS: On the second day after surgery, adrenalectomized rats showed undetectable aldosterone with natriuresis, hyponatremia, decreased urinary potassium excretion and hyperpotassemia. CsA reduced Ccr, induced urinary proteins and up-regulated COL-I, TGF-ß1, CTGF and PAI-1 gene expression with a significant development of TIF. Eplerenone administration prevented TIF and COL-I, TGF-ß1 and PAI-1 up-regulation but did not improve renal function. CONCLUSION: Our results suggest local renal aldosterone is an important mediator of renal injury induced by CsA.

JNK-dependent AP-1 activation is required for aristolochic acid-induced TGF-ß1 synthesis in human renal proximal epithelial cells.

Chronic aristolochic acid nephropathy (CAAN) is a chronic and progressive tubulointerstitial nephropathy characterized by extensive interstitial fibrosis. Aristolochic acid (AA) could induce overexpression of transforming growth factor-ß1 (TGF-ß1) in a human renal proximal tubule epithelial cells line (HKC), which has been implicated in the pathogenesis of CAAN. The present studies in HKC cells showed 1) AA could activate JNK in time- and dose-dependent manners and JNK inhibitor SP600125 could inhibit AA-induced TGF-ß1 promoter activity and TGF-ß1 synthesis; 2) AA-induced JNK activation and TGF-ß1 synthesis were significantly inhibited by kinase-inactive mutants of MEKK4, MKK4, or MKK7; 3) AA could upregulate luciferase activity derived by a wild-type TGF-ß1 promoter, but not by an AP-1 binding-deficient TGF-ß1 promoter; and 4) AA could upregulate expression of c-Fos, phospho-c-Jun, and phospho-ATF2. The above data suggest AA-induced TGF-ß1 overexpression in HKC cells may be mainly mediated by the JNK signaling pathway. Both the upstream kinases of JNK including MEKK4, MKK4, and MKK7, and the downstream transcription factor of JNK, AP-1, may also participate in this process.

[Organic anion transporter system mediates the transport of aristolochic acid in tubular epithelial cells].

OBJECTIVE: To investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism. METHODS: Intracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively. RESULTS: Aristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively. CONCLUSION: Aristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.

[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line].

OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.

Axin is a scaffold protein in TGF-beta signaling that promotes degradation of Smad7 by Arkadia.

TGF-beta signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF-beta pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF-beta signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF-beta signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF-beta signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse-chase experiments further illustrated that Axin significantly decreased the half-life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF-beta signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt-1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF-beta signaling.

[The role of MAPK/ERK1/2 signaling pathway in aldosterone stimulated transforming growth factor-beta1 synthesis in renal tubular epithelial cells].

OBJECTIVE: Our previous study have demonstrated that the expression of transforming growth factor-beta1 (TGF-beta1) by HKC could be up-regulated by aldosterone (ALDO) in vitro. The present study was designed to evaluate the role of MAPK/ERK1/2 phosphorylation in mediating the synthesis of TGF-beta1 in renal tubular epithelial cells that was activated by aldosterone. METHODS: The following tests were performed in vitro: (1) HKC were pretreated with different concentrations of specific ERK1/2, JNK and P38 MAPK pathway inhibitors for 4h, then HKC were stimulated with 10(-7) mol/L ALDO for 48 h, finally enzyme-linked immunosorbent assay (ELISA) were performed to detect TGF-beta1 expression; (2) HKC were stimulated with ALDO at different concentrations and times, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC. (3) HKC which were co-stimulated with 10(-7) mol/L ALDO and different concentrations of spironolactone or specific glucocorticoid hormone receptor inhibitor RU486 for 30min, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC. RESULTS: (1) the production in 15 and 25 micromol/L U0126 incubated groups was (87 +/- 11) pg/ml and (75 +/- 19) pg/ml respectively, which was significantly decreased compared with that in 10(-7) mol/L ALDO incubated group (P < 0.05), however, the amount of TGF-beta1 in these groups were still significant higher than that in the control group (P < 0.05). The production of TGF-beta1 in the groups which were incubated with SP600125 and SB203580 did not appear significant decrease compared with that in 10(-7) mol/L ALDO incubated group (P > 0.05), the production of TGF-beta1 in these groups was also significant higher than that in the control group (P < 0.05). (2) The Phos/Total ERK1/2 ratio was increased in a dose-dependent manner. After HKC were stimulated with 10(-9) - 10(-7) mol/L ALDO for 30 min. Phos/TotalERK1/2 ratio was 0.67 +/- 0.06 and 0.80 +/- 0.05 respectively, which was significantly increased (vs 0 mol/L ALDO, P < 0.05 or 0.01). The expression of Phos/Total ERK1/2 ratio also had a positive correlations with the production of TGF-beta1 (R = 0.793, P < 0.01). With 10(-7) mol/L ALDO stimulated at different times, the Phos/Total ERK1/2 ratio was also significantly increased (vs 0 h, P < 0.05 or 0.01), which began to be increased and reached the peak at 15 min, the relatively ratio was 0.84 +/- 0.06, and waned till 240 min, and returned to normal level at 360 min. (3) After co-stimulated with 10(-7) mol/L ALDO and different concentration of spironolactone, Phos/TotalERK1/2 ratio was significantly decreased along with the concentrations of spironolactone, the relatively ratio of in 10(-9) - 10(-7) was 0.62 +/- 0.08 and 0.60 +/- 0.04 separately (vs 0 mol/L ALDO, or 0.01), but they were still significantly higher than that in control group (P < 0.05). The Phos/TotalERK1/2 ratio was not changed significantly along with the concentrations of RU486 (P > 0.05). CONCLUSION: The effect of aldosterone in up-regulating the expression of TGF-beta1 in HKC is mediated, at least in part, by MAPK/ERK1/2 pathway. Aldosterone may exert this effect on the conditions of binding to the mineralocorticoid receptor first.

Axin stimulates p53 functions by activation of HIPK2 kinase through multimeric complex formation.

Axin and p53 are tumor suppressors, controlling cell growth, apoptosis, and development. We show that Axin interacts with homeodomain-interacting protein kinase-2 (HIPK2), which is linked to UV-induced p53-dependent apoptosis by interacting with, and phosphorylating Ser 46 of, p53. In addition to association with p53 via HIPK2, Axin contains a separate domain that directly interacts with p53 at their physiological concentrations. Axin stimulates p53-dependent reporter transcription in 293 cells, but not in 293T, H1299, or SaOS-2 cells that are defective in p53 signaling. Axin, but not AxindeltaHIPK2, activates HIPK2-mediated p53 phosphorylation at Ser 46, facilitating p53-dependent transcriptional activity and apoptosis. Specific knockdown of Axin by siRNA reduced UV-induced Ser-46 phosphorylation and apoptosis. Kinase-dead HIPK2 reduced Axin-induced p53-dependent transcriptional activity, indicating that Axin stimulates p53 function through HIPK2 kinase activity. Interestingly, HIPK2deltaAxin that lacks its Axin-binding region acts as a dominant-positive form in p53 activation, suggesting that the Axin-binding region of HIPK2 is a putative autoinhibitory domain. These results show that Axin acts as a tumor suppressor by facilitating p53 function through integration of multiple factors.

SUMO-1 modification of the C-terminal KVEKVD of Axin is required for JNK activation but has no effect on Wnt signaling.

Axin is a multifunctional protein, regulating Wnt signaling and the c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) pathway as well as tumorigenesis. In the present study, we found that Axin interacts with three SUMO-1 (small ubiquitin-related modifier) conjugating enzymes 3 (E3), PIAS1, PIASxbeta, and PIASy. The extreme C-terminal six amino acid residues of Axin are critical for the Axin/E3 interaction as deletion of the six residues (AxinDeltaC6) completely abolished the ability of Axin to interact with E3 enzymes. AxinDeltaC6 also failed to activate JNK, although it was intact in both its interaction with MEKK1 and homodimerization. Consistent with the presence of a doublet of the KV(E/D) sumoylation consensus motif at the C-terminal end (KVEKVD), we found that Axin is heavily sumoylated. Deletion of the C-terminal six amino acids drastically reduced sumoylation, indicating that the C-terminal six amino acids stretch is the main sumoylation site for Axin. Sumoylation-defective mutants failed to activate JNK but effectively destabilized beta-catenin and attenuated LEF1 transcriptional activity. In addition, we show that dominant negative Axin mutants blocked PIAS-mediated JNK activation, in accordance with the requirement of sumoylation for Axin-mediated JNK activation. Taken together, we demonstrate that sumoylation plays a role for Axin to function in the JNK pathway.