Youkang Zhang

Detection of the hepatitis C virus antigen in kidney tissue from infected patients with various glomerulonephritis

BACKGROUND Several studies have postulated a causal link between hepatitis C virus (HCV) infection and renal diseases through the induction of cryoglobulinaemia. However, the detection of viral antigens within kidneys of HCV-infected patients has proved to be difficult. We studied a cohort of Chinese HCV-infected patients with various glomerulonephritis (GN) in an attempt to detect HCV antigens within their kidneys. METHODS Twenty-one patients with various GN were found to be serum HCV-antibody positive (seven serum HCV-RNA positive simultaneously), at the time of renal biopsy, from January 2005 to April 2008 in our hospital. A murine monoclonal antibody against the HCV-NS3 protein was employed to detect the HCV antigen using immunohistochemistry and immunogold labelling. Their clinical and pathological data were collected and further analysed. RESULTS The HCV-NS3 antigen was detected in six (6/21, 28.6%) HCV-antibody-positive patients by immunohistochemistry and four out of the six were serum HCV-RNA positive (4/7 in HCV-RNA positive, 57.1%). The HCV antigen mainly displayed a linear or granular deposition along glomerular capillary walls and/or mesangial region. Immunoelectron microscopy showed that the labelling of HCV-NS3 was localized mainly in electronic dense deposits. In the HCV-NS3 detectable patients, three patients were with membranoproliferative glomerulonephritis (MPGN), one with membranous nephropathy, one with IgA nephropathy and one with amyloid nephropathy. The age and urinary protein were significantly greater in HCV-NS3-positive patients than those in HCV-NS3 negative, while serum C3 level was significantly lower in the former group. No significant difference was found in serum ALT, albumin and creatinine level between the two groups. CONCLUSION HCV-NS3 antigens could be detected in kidney tissue of HCV-infected patients with various GN, but mainly in those with MPGN and HCV-RNA positive. HCV itself might be involved directly in the pathogenesis of HCV-associated GN.

The clinical and pathological characteristics of Chinese elderly patients with anti-neutrophil cytoplasmic autoantibodies associated small vessel vasculitis

BACKGROUND Anti-neutrophil cytoplasmic autoantibodies (ANCA) are serological markers of ANCA-associated small vessel vasculitis (AASV) which is one of common autoimmune diseases in Caucasian elderly population. OBJECTIVE To analyze the clinical and pathological characteristics of Chinese elderly patients with AASV. METHODS One-hundred forty one Chinese patients with AASV over 65 years old, diagnosed between 1997 and 2001 in the Institute of Nephrology of Peking University First Hospital, were retrospectively studied and their clinical and pathological data were analyzed. RESULTS Patients diagnosed with AASV patient increased chronologically with the yearly ratio in 2000 and 2001 significantly (P < 0.05) higher than that in 1998 and before. Of the 141 patients, 72 were male and 69 were female with an average age of 68.2 years. 13 of the 141 were cytoplasmic ANCA (cANCA) positive and all recognized proteinase 3 (PR3). The other 128 were perinuclear ANCA (pANCA) positive and 120/128 recognized MPO, 8/128 recognized both PR3 and MPO. Less than 50% of the patients were correctly diagnosed within 3 months. Clinically, 78% of the patients had fever and fatigue, 52.5% had body weight loss, 96.4% had kidney involvement, of which 75% had elevated serum creatinine and 30.8% had acute renal failure. 76.6% had lung involvement, over half of them had hemoptysis or lung infiltrates. Other clinical manifestations included arthralgia (48.2%), muscle pain (39.7%), gastrointestinal symptoms (39.7%), eye involvement (28.3%) and ENT involvement (31.2%). In laboratory examinations, 94.4% of the patients had anemia, 62.4% had increased WBC count, 93.6% had increased ESR and 55.1% had increased CRP. CONCLUSIONS More and more patients with AASV were diagnosed in Chinese elderly. Kidney was the most vulnerable organ to be involved and lung was the most important extra-renal organ to be affected. For elderly patients with multi-organ damage, an ANCA test should be performed in order to make an early diagnosis and start therapy in time.

Autoantibodies against glomerular mesangial cells and their target antigens in lupus nephritis.

Mesangial proliferation and deposition of immunoglobulins and complement components within glomerular mesangium was one of the important pathological features of lupus nephritis. Autoantibodies against human mesangial cells could be detected in the sera of patients with IgA nephropathy (IgAN) and Henoch-Schöenlein nephritis. We speculated that autoantibodies against human glomerular mesangial cells might play a role in the development of lupus nephritis. Objective. To screen autoantibodies against human glomerular mesangial cells in sera from patients with lupus nephritis and to identify their target antigens. Methods. Sera were collected from 96 patients with lupus nephritis as well as 25 patients with IgAN and 20 patients with idiopathic membranous nephropathy (IMN). Cell lysates of in vitro cultured human glomerular mesangial cells were used as antigens in Western-blot analysis to detect autoantibodies against human mesangial cells in sera from patients with lupus nephritis as well as IgAN and IMN. The clinical and pathological significance of the autoantibodies were further investigated. Results. Autoantibodies against human mesangial cells could be detected in 94/96 (97.9%) of the sera from patients with lupus nephritis in Western-blot analysis. Twelve protein bands could be blotted by the sera from patients with lupus nephritis. The prevalence of autoantibodies against human mesangial cells in IgAN was 14/25 (56.0%) and only seven protein bands could be blotted. Five autoantibodies (anti-18, 24, 36, 46, and 91 kD) could be detected only in sera from patients with lupus nephritis. In patients with lupus nephritis, some autoantibodies might have some relationship with gender, hematuria, ANA, anti-dsDNA or anti-ENA antibodies. Conclusions. There are autoantibodies directly against heterogeneous antigens of human glomerular mesangial cells in sera from patients with lupus nephritis, and some of them might be associated with different clinical manifestations.

A clinicopathological analysis in a large cohort of Chinese patients with renal amyloid light-chain amyloidosis

BACKGROUND The aim of the study is to investigate the association between clinical and pathological features in a large cohort of Chinese patients with renal immunoglobulin light-chain amyloidosis (AL). METHODS A series of 186 patients with renal AL amyloidosis diagnosed between 1990 and 2011 were retrospectively reviewed. The extent of amyloid deposition in glomeruli, blood vessels and tubulointerstitium were evaluated semiquantitatively. The renal amyloid load was defined by the sum of glomerular, vascular and interstitial deposits. The associations between the clinical manifestations and pathological features were analyzed. RESULTS The extent of glomerular amyloid deposition was positively correlated with the level of proteinuria. Patients with codeposition of amyloid and immune complexes (ICs) in glomeruli had higher levels of proteinuria than those without ICs. Advanced glomerular amyloid deposition was an independent pathological factor associated with renal insufficiency at diagnosis. The degree of vascular amyloid (VA) deposition was positively correlated with cardiac involvement and hepatic involvement. Patients with AL-κ showed a higher prevalence of hepatic involvement and more severe VA deposition than patients with AL-λ. High renal amyloid load independently predicted the increased risk for overall death after adjusting for recognized confounders. CONCLUSIONS The degree and localization of amyloid deposits in the kidney of AL patients were associated with the degree of proteinuria and renal insufficiency, as well as extrarenal organs involvement. There were some differences between AL-κ and AL -λ in clinical and pathological characteristics. The renal amyloid load was an independent predictor for overall mortality.

Ultrastructural Features and Expression of Cytoskeleton Proteins of Podocyte from Patients with Minimal Change Disease and Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) have been suggested for the category of podocytopathies. An ultrastructural observation and immunogold labeling for cytoskeleton proteins of podocytes on 11 cases each of FSGS and MCD were performed. Compared to MCD, more severe ultrastructural alterations of podocyte were identified in FSGS, which were characterized by higher frequency of mat-like condensation of microfilaments in the foot process and the detachment of the foot process from glomerular basement membrane. The labeling of α-actinin of podocytes in FSGS was significantly higher than MCD, which suggested an abnormal expression of cytoskeleton protein of podocyte in FSGS. The present study demonstrated a much more severe podocyte injury at the ultrastructural level in FSGS than in MCD.

Antineutrophil cytoplasmic autoantibodies in patients with systemic lupus erythematosus recognize a novel 69 kDa target antigen of neutrophil granules.

Objective:  Antineutrophil cytoplasmic autoantibodies (ANCA) were found in patients with systemic lupus erythematosus (SLE). Cathepsin G and lactoferrin were the major target antigens. However, some ANCA‐positive sera did not recognize either of them. The present study was to investigate the unknown target antigens of ANCA in patients with SLE and their clinical significance.

Diffuse thin glomerular basement membrane in association with Fabry disease in a Chinese female patient

We report a 41-year-old Chinese female with Fabry disease and diffuse thinning of the glomerular basement membrane (GBM). The patient presented with peripheral edema, mild proteinuria, microscopic hematuria, normal renal function, hypertension and tinnitus. Family screening showed that her daughter had microscopic hematuria, tinnitus and neuropathic pain. Renal biopsy of the proband showed focal segmental glomerulosclerosis with cytoplasmic vacuolization of the glomerular visceral epithelial cells by light microscopy. Laminated myelin inclusions in some of the glomerular podocytes, parietal epithelia, distal tubular epithelial cells and vascular endothelial cells along with diffuse thinning of the GBM (mean thickness of GBM: 216 ± 31 nm) were identified by electron microscopy. Genetic analysis detected a de novo novel GLA mutation, 1208 ins 21 bp, while a new variant of COL4A3 SNP M1209I was carried by mother and daughter as well as the probands father (I-1) and one sister (II-4). The coexistence of thinned GBM should be considered in patients with Fabry disease-manifested familial hematuria.

The Effects of Indoxyl Sulfate on Human Umbilical Cord-Derived Mesenchymal Stem Cells In Vitro

Background/Aims: Indoxyl sulfate, an important protein-bound uremic toxin, can damage stem cells, thus hampering stem cell-based regenerative medicine approaches targeting chronic kidney diseases (CKD). Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are thought to have promising clinical application because of their high proliferative potential and ease of isolation than MSCs from other sources. In the present study, we aimed to determine the harmful effects of indoxyl sulfate on the phenotype and functional potential of hUC-MSCs in vitro. Methods: The toxicity and cell viability was examined by Trypan blue exclusion and MTT assay. The cellular surface markers and the percentage of apoptotic cells by Annexin-V/PI staining were analyzed by flow cytometry. Proliferation was evaluated based on cell number counting and Ki-67 immunostaining. Cell senescence was measured using senescence-associated β-Galactosidase activity. The ability to stimulate the development of CD4+CD25+FoxP3+ regulatory T cells was assessed by incubating hUC-MSCs with peripheral blood mononuclear cells from the healthy volunteers. Results: Our results demonstrated that the immunophenotype of hUC-MSCs was not affected by indoxyl sulfate flow cytometry. However, a significant decrease in cell numbers and fraction of Ki-67 positive proliferating cells, along with a significant increase in cellular senescence were detected in hUC-MSCs after exposure to indoxyl sulfate. Additionally, their ability to stimulate CD4+CD25+FoxP3+ regulatory T cell production was compromised when hUC-MSCs were pretreated with indoxyl sulfate. Conclusion: Taken together, our study clearly demonstrated that the molecular alterations and functional incompetence in hUC-MSCs under the challenge of indoxyl sulfate in vitro.

Simultaneous occurrence of hepatitis C virus-associated glomerulonephritis and AL amyloidosis

We report a 65-year-old male with hepatitis C virus (HCV)-associated glomerulonephritis (GN) and AL amyloidosis. This patient had a 14-year history of HCV infection with positive serum HCV RNA at presentation. Diagnosis of HCV-related GN was established using immunohistochemistry in which the HCV-NS3 antigen was mainly detected as granular deposition in glomerular mesangium. AL amyloidosis was determined by electron microscopy, positive Congo red staining and identification of lambda-chain in the kidney specimen and monoclonal IgG-lambda in serum and urine. Under immunoelectron microscopy, the HCV-NS3 antigen was found in electron-dense deposits, while lambda-chains appeared in the amyloid-like filaments.

Low-dose IL-2 expands CD4+ regulatory T cells with a suppressive function in vitro via the STAT5-dependent pathway in patients with chronic kidney diseases

Abstract Background: Patients with chronic kidney disease (CKD) often have CD4+ regulatory T cells (Tregs) dysfunction and chronic inflammation. We aim to investigate the effect, function, and related mechanism of low-dose IL-2 on CD4+ regulatory T cells expansion in vitro from patients with CKD. Methods: A total of 148 newly diagnosed patients with CKD at Stage III and 35 healthy volunteer subjects were recruited into our studies. The number of peripheral Tregs in peripheral blood mononuclear cells isolated from CKD patients, which were characterized by FACS as CD4+CD25hi and CD4+CD25+FoxP3+. The effect of low-dose IL-2 on expansion of Tregs, and the suppressive function of expanded Tregs were also analyzed by FACS. The levels of FoxP3 mRNA were detected by qRT-PCR. The activation of IL-2 induced Stat5 and blocking experiments were assessed by Western Blotting and FACS. Results: We found that the frequency of peripheral Tregs from CKD patients was significantly lower than that in healthy volunteer subjects. We also showed that IL-2 selectively expanded CD4+CD25hi and CD4+CD25+FoxP3+ regulatory T cells, and also upregulated the expression of FoxP3 mRNA. Our in vitro studies demonstrated that expanded CD4+ regulatory T cells from CKD patients suppressed proinflammatory Th1 and Th17 cell response. Furthermore, STAT5 activation is required for IL-2-induced expansion of regulatory T cells and expression of FoxP3 mRNA from CKD patients. Conclusions: Our findings support the clinical Treg defects in CKD patients with glomerular diseases, and the rationale of evaluating low-dose IL-2 treatment for selectively modulating CD4+ Tregs.