Wenfang Chen

A Crosstalk between the Smad and JNK Signaling in the TGF-β-Induced Epithelial-Mesenchymal Transition in Rat Peritoneal Mesothelial Cells

Transforming growth factor β (TGF-β) induces the process of epithelial-mesenchymal transition (EMT) through the Smad and JNK signaling. However, it is unclear how these pathways interact in the TGF-β1-induced EMT in rat peritoneal mesothelial cells (RPMCs). Here, we show that inhibition of JNK activation by introducing the dominant-negative JNK1 gene attenuates the TGF-β1-down-regulated E-cadherin expression, and TGF-β1-up-regulated α-SMA, Collagen I, and PAI-1 expression, leading to the inhibition of EMT in primarily cultured RPMCs. Furthermore, TGF-β1 induces a bimodal JNK activation with peaks at 10 minutes and 12 hours post treatment in RPMCs. In addition, the inhibition of Smad3 activation by introducing a Smad3 mutant mitigates the TGF-β1-induced second wave, but not the first wave, of JNK1 activation in RPMCs. Moreover, the inhibition of JNK1 activation prevents the TGF-β1-induced Smad3 activation and nuclear translocation, and inhibition of the TGF-β1-induced second wave of JNK activation greatly reduced TGF-β1-induced EMT in RPMCs. These data indicate a crosstalk between the JNK1 and Samd3 pathways during the TGF-β1-induced EMT and fibrotic process in RPMCs. Therefore, our findings may provide new insights into understanding the regulation of the TGF-β1-related JNK and Smad signaling in the development of fibrosis.

Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway

Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes.

Up-regulated renal expression of TNF-α signalling adapter proteins in lupus glomerulonephritis

The role of tumour necrosis factor (TNF-α) signalling adapters in lupus nephritis (LN) is poorly understood. This study investigated renal expression of TNF-α and TNF signalling adapter proteins, including TNF receptor-associated death domain protein (TRADD), receptor-interacting protein (RIP) and TNF receptor-associated factor-2 (TRAF-2) in patients with LN. The renal expression of proliferating cell nuclear antigen (PCNA) and CD68 was also measured. The study showed that glomerular and tubular expression of TNF-α, TRADD, RIP and TRAF-2 was significantly up-regulated in class III and IV LN in which the intense staining was observed on the crescents, proximal and distal tubules and interstitial mononuclear cells. The number of PCNA-positive cells and CD68-positive cells (macrophages) was increased obviously in class III and IV LN. There was a correlation between the expression levels of TNF-α, TRADD, RIP, TRAF-2 and the number of PCNA-positive or CD68-positive cells and active index of renal pathology. These findings suggest that TNF-α and TNF-α adapters in patients with LN play a role in immunopathogenic injury via transmitting abnormal cell proliferating and proinflammatory signals. The findings have provided further insights into the role of TNF-α and its adapter proteins in the pathogenesis of LN and have important therapeutic implications.

Identification of apolipoprotein E Guangzhou (arginine 150 proline), a new variant associated with lipoprotein glomerulopathy

Background/Aims: Lipoprotein glomerulopathy (LPG) is a rare disease characterized by thrombus-like substances in markedly dilated glomerular capillaries and elevated plasma levels of apolipoprotein E (apoE). Previous studies have shown that genetic disorders of apoE may contribute to the pathogenesis of LPG, but LPG may not be caused by apoE gene mutations in Chinese patients. This study investigated the association of a new variant of apoE with LPG in a Chinese family. Methods: The apoE gene in a family with 4 LPG patients was sequenced. The polymerase chain reaction product of coding region of apoE exon 4 was cloned into pMD 18-T vector and then sequenced. Results: A novel point mutation in exon 4 of the apoE gene was identified in all 4 LPG patients and 1 asymptomatic family member. Sequence analysis confirmed a nucleotide G to C point mutation in exon 4 (base 308) of the apoE gene in all patients and the asymptomatic family member. This missense mutation denotes amino acid substitution of the proline residue for arginine residue at position 150 of apoE. Those patients were all heterozygotes with apoE Guangzhou. One of 2 grandsons was a heterozygous carrier of apoE Guangzhou, although he did not have proteinuria. Conclusion: The results of this study suggest that apoE (arginine 150 proline) is a novel apoE variant that etiologically related to LPG. This variant (apoE Guangzhou) may cause a marked molecular conformational change of the apoE and thus impair its binding ability to lipids.

Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis.

Objective Several studies have reported that tacrolimus (TAC) significantly reduced proteinuria in lupus nephritis (LN) patients and mouse models. However, the mechanism for this effect remains undetermined. This study explored the mechanism of how TAC protects podocytes from injury to identify new targets for protecting renal function. Methods MRL/lpr mice were given TAC at a dosage of 0.1 mg/kg per day by intragastric administration for 8 weeks. Urine and blood samples were collected. Kidney sections (2μm) were stained with hematoxylin-eosin (HE), periodic acid-Schiff base (PAS) and Massons trichrome stain. Mouse podocyte cells (MPC5) were treated with TAC and/or TGF-β1 for 48h. The mRNA levels and protein expression of synaptopodin and Wilms’ tumor 1 (WT1) were determined by real-time PCR, Western blotting and/or immunofluorescence, respectively. Flow cytometry was used to detect cell apoptosis with annexin V. Podocyte foot processes were observed under transmission electron microscopy. IgG and C3 deposition were assessed with immunofluorescence assays and confocal microscopy. Results Synaptopodin expression significantly decreased in MRL/lpr disease control mice, accompanied by increases in 24-h proteinuria, blood urea nitrogen, and serum creatinine. TAC, however, reduced proteinuria, improved renal function, attenuated renal pathology, restored synaptopodin expression and preserved podocyte numbers. In MPC5 cells, TGF-β1 enhanced F-actin damage in podocytes and TAC stabilized it. TAC also decreased TGF-β1-induced podocyte apoptosis in vitro and inhibited foot process fusion in MRL/lpr mice. In addition, our results also showed TAC inhibited glomerular deposition of IgG and C3. Conclusion This study demonstrated that TAC reduced proteinuria and preserved renal function in LN through protecting podocytes from injury partly by stabilizing podocyte actin cytoskeleton and inhibiting podocyte apoptosis.

Impaired TGF-β signalling enhances peritoneal inflammation induced by E. Coli in rats

BACKGROUND Peritonitis is a common and severe complication of peritoneal dialysis (PD). Although TGF-beta is a key mediator in peritoneal fibrosis with chronic PD, its role in acute peritoneal inflammation remains unclear. METHODS Potential role of TGF-beta signalling in acute peritonitis was investigated in a rat model by infecting peritoneum with E. coli and in primary culture of peritoneal mesothelial cells (PMC) by LPS. RESULTS We found that a single infection of E. coli caused an acute, but transient peritonitis by a significant increase in ascites white blood cells (WBC), peritoneal CD45+ leukocytes, upregulation of TNFalpha, activation of NF-kappaB/p65 and impaired peritoneal function (all P < 0.01). Interestingly, spontaneous recovery of acute peritonitis occurred with upregulation of TGF-beta1 and activation of Smad2/3, suggesting a protective role of TGF-beta signalling in acute peritonitis. This was demonstrated by the finding that blockade of the TGF-beta signalling pathway with gene transfer of Smad7 inactivated peritoneal Smad2/3 but worsened E. coli-induced, NF-kappaB-dependent peritoneal inflammation and peritoneal dysfunction (all P < 0.01). Furthermore, studies in vitro also found that impaired TGF-beta signalling by overexpressing Smad7 in PMC were able to overcome the inhibitory effect of TGF-beta on LPS-induced, NF-kappaB-mediated peritoneal inflammation. CONCLUSION Results from this study demonstrate that TGF-beta signalling is essential in protection against acute peritoneal inflammation induced by bacterial infection.

Influence of dialysis modality on renal transplant complications and outcomes.

AIMS The present study investigated the influence of the pretransplant dialysis modality, hemodialysis (HD) or peritoneal dialysis (PD), on renal transplant complications and outcomes. METHODS 402 cadaveric renal transplant patients maintained on HD (N = 303) or PD (N = 99) for more than 3 months prior to transplantation were studied retrospectively, and a total of 345 patients were followed up for 30.2 +/- 15.2 months. The impact of HD or PD on acute rejection, delayed graft function (DGF), infection, chronic rejection, and the survival rate of graft and patients were analyzed. RESULTS There was no significant difference between the HD and PD groups with regard to the causes of end-stage renal disease, age, gender, blood pressure, hemoglobin, HLA match, hot and cold ischemia time, and hepatitis C virus infection. The incidence rates of DGF, acute rejection, chronic rejection and cytomegalovirus and other infections were also not significantly different between the HD and PD groups. However, compared to HD, patients with PD had longer dialysis duration (p < 0.05), but less hepatitis B infection (p < 0.05) and post-transplant infection (p < 0.05). In contrast, in those PD patients with hepatitis B infection, graft loss was significantly increased (19.23% vs. 8.86% , p = 0.021). The incidence of acute rejection episodes was higher in HD patients who had pretransplant dialysis for more than 12 months (p < 0.05). The overall patient and graft survival rates within 5 years between the HD and PD groups were not significantly different (p > 0.05). CONCLUSIONS The influence of PD and HD on complications after renal transplant at 1 year and 5 years and graft survival rates was similar, and therefore, either HD or PD can be chosen as the pretransplant dialysis modality. However, patients in the PD group had a reduced incidence of hepatitis virus infection, suggesting that PD may have certain advantages over HD as a preoperative substitution therapy for renal transplantation.

V-ATPase Promotes Transforming Growth Factor-β-induced Epithelial-Mesenchymal Transition of Rat Proximal Tubular Epithelial Cells

The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.

Clinicopathologic features and treatment response in nephrotic IgA nephropathy with minimal change disease.

OBJECTIVE To analyze the clinicopathological features and therapeutic response of nephrotic IgA nephropathy (IgAN) patients with minimal-change disease (MCD). METHODS 62 nephrotic IgAN patients were enrolled between January 2002 and December 2008, and divided into two groups including Group A: patients with MCD-like pathological features, and Group B with non-MCD pathologic pattern. The clinicopathological features, treatments, and responses were then analyzed. RESULTS 13 (21.0%) patients exhibited MCD-like pathological changes. Patients in Group A presented more prominent proteinuria, hypoalbuminemia but higher hemoglobin and no incidence of renal insufficiency compared to Group B (p < 0.05). 62 patients were treated with corticosteroid, and the complete remission rate in Group A is higher than that in Group B (84.6% vs. 34.7%, p = 0.008), but the relapse rate is much higher in Group A (53.8% vs. 20.4%, p = 0.03). 21 patients were treated combining with immunosuppressant due to unresponsiveness or relapse, which yielded a high re-remission rate in Group A (100%). After follow-up of 53.9 ± 26.9 months, the 5-year renal survival rate is higher in Group A (100%) than that in Group B (84.7%), but no significant difference was observed (p = 0.24). CONCLUSIONS MCD-like pathological changes exist in quite a few nephrotic IgAN patients. These IgAN patients responded well to corticosteroid monotherapy but had a higher rate of relapse. Though manifesting with severe nephrotic symptoms, they tended to have a favorable clinical outcome probably due to the minimal pathological changes. Nevertheless, larger sample size and longer follow-up periods are needed for better understanding of the disease.

Clinical outcomes of lupus nephritis patients with different proportions of crescents.

Background The prognostic significance of different proportions of crescents in lupus nephritis (LN) remains unclear. We assessed the long-term prognosis of LN patients with different proportions of crescents. Methods In this single-center, retrospective cohort study, 788 eligible LN patients were enrolled. The primary endpoint was doubling of baseline serum creatinine and end-stage renal disease; the secondary endpoint was death. Results There were 406 (51.5%) patients demonstrating crescents at biopsy, and they had more severe baseline status: lower estimated glomerular filtration rate (eGFR), more proteinuria, more severe microscopic hematuria, and higher pathological scores for both activity index (AI) and chronicity index (CIn) (all p < 0.001, respectively). After a median follow-up period of 56 months (range: 3–172 months), no significant differences were observed in terms of renal or patient survival in these two groups (p = 0.188). In LN patients with crescents, patient survival was poorer along with the increase in the proportion of crescents; for crescentic LN, patient survival was 78.9% at five years and 52.6% at 10 years (vs. subgroups of crescent proportion <10%, 10%–19%, 20%–49%: 95.5%, 92.3%, 91.7% at five years; 86.1%, 80.1%, 66.5% at 10 years, respectively, p = 0.008). Furthermore, there were higher proportions of crescents independently predicted for adverse outcomes of renal progression and mortality (p = 0.049) after adjusting for age, sex, baseline eGFR, proteinuria, AI, and CIn in the multivariate Cox proportional hazard model. Conclusion The overall long-term renal survival of LN patients with and without crescents was comparable. However, a higher proportion of crescents increased the risk for unfavorable outcomes. Therefore, more attention should be paid to the lesions of crescents, and more prospective studies are needed to explore the optimal regimens for LN patients with different proportions of crescents.