Zhaohong Chen

Anti-Phospholipase A2 Receptor Antibody in Membranous Nephropathy

The M-type phospholipase A2 receptor (PLA2R) is a target autoantigen in adult idiopathic membranous nephropathy (MN), but the prevalence of autoantibodies against PLA2R is unknown among Chinese patients with MN. Here, we measured anti-PLA2R antibody in the serum of 60 patients with idiopathic MN, 20 with lupus-associated MN, 16 with hepatitis B (HBV)-associated MN, and 10 with tumor-associated MN. Among patients with idiopathic MN, 49 (82%) had detectable anti-PLA2R autoantibodies using a Western blot assay; an assay with greater sensitivity detected very low titers of anti-PLA2R in 10 of the remaining 11 patients. Using the standard assay, we detected anti-PLA2R antibody in only 1 patient with lupus, 1 with HBV, and 3 with cancer, producing an overall specificity of 89% in this cohort limited to patients with secondary MN. The enhanced assay detected low titers of anti-PLA2R in only 2 additional samples of HBV-associated MN. In summary, these results suggest that PLA2R is a major target antigen in Chinese idiopathic MN and that detection of anti-PLA2R is a sensitive test for idiopathic MN.

Influence of CYP3A5 and MDR1 polymorphisms on tacrolimus concentration in the early stage after renal transplantation

Abstract:  Objective:  Tacrolimus is an immunosuppressive drug with a narrow therapeutic range and wide interindividual variation in its pharmacokinetics. Cytochrome P450 (CYP) 3A and P‐glycoprotein (P‐gp, encoded by MDR1) play an important role in the absorption and metabolism of tacrolimus. The objective of this study was to evaluate whether or not CYP3A5*1/*3 or MDR1 C3435T polymorphisms are associated with the tacrolimus concentration per dose.

Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.

Downregulation of MicroRNA-30 Facilitates Podocyte Injury and Is Prevented by Glucocorticoids

MicroRNAs (miRNAs) are essential for podocyte homeostasis, and the miR-30 family may be responsible for this action. However, the exact roles and clinical relevance of miR-30s remain unknown. In this study, we examined the expression of the miR-30 family in the podocytes of patients with FSGS and found that all members are downregulated. Treating cultured human podocytes with TGF-β, LPS, or puromycin aminonucleoside (PAN) also downregulated the miR-30 family. Podocyte cytoskeletal damage and apoptosis caused by treatment with TGF-β or PAN were ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. Moreover, we found that miR-30s exert their protective roles by direct inhibition of Notch1 and p53, which mediate podocyte injury. In rats, treatment with PAN substantially downregulated podocyte miR-30s and induced proteinuria and podocyte injury; however, transfer of exogenous miR-30a to podocytes of PAN-treated rats ameliorated proteinuria and podocyte injury and reduced Notch1 activation. Finally, we demonstrated that glucocorticoid treatment maintains miR-30 expression in cultured podocytes treated with TGF-β, LPS, or PAN and in the podocytes of PAN-treated rats. Glucocorticoid-sustained miR-30 expression associated with reduced Notch1 activation and alleviated podocyte damage. Taken together, these findings demonstrate that miR-30s protect podocytes by targeting Notch1 and p53 and that the loss of miR-30s facilitates podocyte injury. In addition, sustained miR-30 expression may be a novel mechanism underlying the therapeutic effectiveness of glucocorticoids in treating podocytopathy.

Triptolide reduces proteinuria in experimental membranous nephropathy and protects against C5b-9-induced podocyte injury in vitro

Membranous nephropathy is a major cause of nephrotic syndrome in adults where podocyte injuries were found to mediate the development of proteinuria. Triptolide, a major active component of Tripterygium wilfordii Hook F, has potent immunosuppressive, anti-inflammatory and antiproteinuric effects. To study its antiproteinuric properties, we established an experimental rat model of passive Heymann nephritis and a C5b-9 injury model of podocytes in vitro. Treatment or pretreatment with triptolide markedly reduced established proteinuria as well as the titer of circulating rat anti-rabbit IgG antibodies in these nephritic rats, accompanied by a reduction in glomerular C5b-9 deposits. Expression of desmin, a marker of podocyte injury, diminished after triptolide treatment, whereas quantitative analysis of mean foot process width showed that effacement of foot processes was substantially reversed. In in vitro studies we found that triptolide deactivated NADPH oxidase, suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase, and restored RhoA signaling activity. Triptolide did not interfere with the formation of C5b-9 on the membrane of podocytes. Thus, triptolide reduces established heavy proteinuria and podocyte injuries in rats with passive Heymann nephritis, and protects podocytes from C5b-9-mediated injury.

Evaluation of podocyte lesion in patients with diabetic nephropathy: Wilms' tumor-1 protein used as a podocyte marker

INTRODUCTION The reduction of podocyte number and density per glomerulus has been linked to the development of proteinuria and the progression of disease in patients with diabetic nephropathy (DN). However, it has been recognized that measurement of podocyte number by light microscope is quite difficult because of the complexity of both podocyte and glomerular structure, which is not suitable for clinical research. In our research institute, we used WT1 as podocyte marker to evaluate the podocyte lesion. METHODS In our experiment, we selected the C-terminal antibody of WT1 to stain the nuclei and the N-terminal antibody of WT1 to stain the cytoplasma of podocytes. Forty patients were enrolled with type 2 diabetes and proven to have DN by renal biopsy analysis. DN patients were classified into three groups based on the degree of proteinuria: microalbuminuria (n=10, 30-300mg/24h), overt proteinuria (n=15, 0.5-3.5g/24h), and heavy proteinuria (n=15, >3.5g/24h). RESULTS The results demonstrated that the podocyte number was markedly decreased in patients with DN (30-51% reduction). There was a significant negative correlation between the proteinuria and both podocyte density and number. The cover area density of podocyte cytoplasma in glomerulus was also significantly decreased in all DN patients (39-80% reduction). A significant inverse correlation was observed between the cover area density and the degree of proteinuria. The correlation coefficient (r=-0.85) was much higher than that between proteinuria and podocyte density (r=-0.56) or podocyte number (r=-0.36). CONCLUSION In conclusion, podocyte damage occurred in patients with DN, even in the early stage and became more dramatic during the course of proteinuria progression. WT1 staining, using the polyclonal antibody to stain the nuclei and monoclonal antibody to stain the cytoplasma of podocytes together, is a valuable alternative technique in the study of podocyte injury.

Podocyte autophagic activity plays a protective role in renal injury and delays the progression of podocytopathies.

The progression of podocytopathies is quite variable among patients and the underlying reason for this remains unclear. Here, we report that autophagic activity in podocytes plays a critical role in controlling the progression of podocytopathies. Morphological and biochemical studies on renal biopsies from patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS) showed that glomeruli, and in particular podocytes, from MCD patients had higher levels of Beclin1‐mediated autophagic activity than glomeruli from FSGS patients. Repeat renal biopsies of MCD patients enabled tracking of podocyte autophagic activity and confirmed that patients maintaining high podocyte autophagic activity retained MCD status, whereas patients with decreased podocyte autophagic activity progressed to FSGS. Inhibition of autophagic activity, by knocking down Beclin1 or by treating with 3‐methyladenine (3‐MA) or chloroquine, enhanced puromycin aminonucleoside (PAN)‐induced apoptosis of podocytes. In contrast, rapamycin‐mediated promotion of autophagic activity decreased this apoptosis. In PAN‐treated rats, inhibition of autophagy with 3‐MA or chloroquine resulted in earlier onset and greater proteinuria, more extensive foot‐process effacement, and reduction in podocyte markers, whereas rapamycin‐mediated stimulation of autophagy led to decreased proteinuria and less severe foot‐process effacement, but higher expression of podocyte markers. This study demonstrates that podocyte autophagic activity plays a critical protective role in renal injury and that maintaining podocyte autophagic activity represents a potential therapeutic strategy for controlling the progression of podocytopathies. Copyright

Improvement of immune dysfunction in patients with severe acute pancreatitis by high-volume hemofiltration: a preliminary report.

Objective The aim of this study was to investigate the effect of high-volume hemofiltration (HVHF) on ameliorating immune dysfunction in patients with severe acute pancreatitis (SAP). Methods Twelve patients diagnosed with SAP admitted to the intensive care unit of general surgery, Jinling Hospital, from January 2004 to December 2006 were included in this study. They were assigned to the standard medical therapy group (SMT group, n=4) or HVHF group (n=8) immediately after enrollment, in a 1:2 ratio. The SMT group were given standard treatment for SAP, while the HVHF group were given standard as well as 72-hour HVHF treatment initiated within 2 hours after enrollment. Patients in the 2 groups were comparable for the baseline clinical parameters. All patients were monitored over a 72-hour observation period for continuous clinical status, blood cell counts including monocytes, CD4+ and CD8+ T cells, and HLA-DR expression on monocytes. Blood samples were collected from those patients at 0, 6, 12, 24, 48, and 72 hour after enrollment for measurement of plasma Th1-type cytokines (interleukin-1 [IL-1], IL-2, interferon-γ [IFN-γ], and tumor necrosis factor-α [TNF-α]) and Th2-type cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) using ELISA. Results Within 72 hours, all measured cytokines except IL-4 were maintained at high levels, accompanied with a low level of peripheral monocytes, CD4+ and CD8+ T cell counts, and HLA-DR expression. Seventy-two hours later, plasma cytokines IFN-γ, IL-1, IL-2, IL-5, IL-10, and IL-13 (p<0.05), but not TNF-α and IL-6, in patients in the HVHF group were significantly reduced, while there was no change for these parameters in the SMT group. Plasma levels of IFN-γ, TNF-α, IL-1, IL-2, IL-5, and IL-13 in the HVHF group were significantly lower than those in the SMT group. Peripheral CD4+ and CD8+ T cells, monocyte count, and HLA-DR expression were increased significantly (p<0.05) only in the HVHF group, not in the SMT group. HLA-DR expression in the HVHF group was significant higher than that in the SMT group (p<0.05). Conclusions HVHF significantly reduced plasma inflammatory cytokine concentrations including those of IFN-γ, TNF-α, IL-1, IL-2, IL-5, and IL-13, while it increased monocyte HLA-DR expression in patients with SAP. The association of plasma cytokine reduction and cellular immune function recovery and clinical outcome needs further investigation.

Toll-like Receptor 9 Can be Activated by Endogenous Mitochondrial DNA to Induce Podocyte Apoptosis.

Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases.

Predominance of Intraglomerular T-bet or GATA3 May Determine Mechanism of Transplant Rejection

The transcription factors T-bet and GATA3 determine the differentiation of helper T cells into Th1 or Th2 cells, respectively. An altered ratio of their relative expression promotes the pathogenesis of certain immunological diseases, but whether this may also contribute to the pathogenesis of antibody-mediated rejection (ABMR) versus T cell-mediated rejection (TCMR) is unknown. Here, we characterized the intragraft expression of T-bet and GATA3 and determined the correlation of their levels with the presence of typical lesions of ABMR and TCMR. We found a predominant intraglomerular expression of T-bet in patients with ABMR, which was distinct from that in patients with TCMR. In ABMR, interstitial T-bet expression was typically located in peritubular capillaries, although the overall quantity of interstitial T-bet was less than that observed in TCMR. The expression of intraglomerular T-bet correlated with infiltration of CD4+ and CD8+ lymphocytes, which express T-bet, as well as intraglomerular CD68+ monocyte/macrophages, which do not express T-bet. The predominance of intraglomerular T-bet expression relative to GATA3 expression associated with poor response to treatment with bolus steroid. In summary, predominance of intraglomerular T-bet expression correlates with antibody-mediated rejection and resistance to steroid treatment.