1. Increased mesangial cell hyaluronan expression in lupus nephritis is mediated by anti- DNA antibody-induced IL-1β 
S. Yung, R.C.W. Tsang, J.K.H. Leung, T.M. Chan 
Kidney International 
Volume 69, Issue 2, Pages (January 2006)
DOI: /sj.ki
Copyright © 2006 International Society of Nephrology Terms and Conditions

2. Figure 1
Representative renal HA expression. Paraffin sections of normal tissue obtained from patients who underwent nephrectomy for tumor (a, d, g, and j) or renal biopsies from patients with diffuse proliferative LN (b, c, e, f, h, i, k, and l) were subjected to staining with eosin and hematoxylin (a–c). Sections were also used for cytochemical staining for HA expression using the peroxidase–anti-peroxidase method (d–i). Immunoreactive products were visualized using diaminobenzidine as chromagen and counterstained with hematoxylin. Constitutive HA expression was observed in the periglomerular tissue and tubules but not within the glomerulus in control specimens (d). Specimens obtained from LN patients demonstrated increased HA expression in the glomerulus (asterisk), in addition to increased periglomerular and tubular staining (e and f). In 25% of LN biopsies, increased HA expression was also observed in infiltrating cells in the glomerulus (f, arrow). Part of the glomerulus (▭) shown in d–f is magnified to facilitate comparison of HA expression in control and LN renal tissues (g–i). Cryosections were used to determine IgG deposition within the glomeruli (j–l). Original magnification × 200 (a–f) and (j–l). Original magnification × 1600 (g–i). The expression of IgG deposition and HA in the glomerulus were scored without knowledge of other clinical or laboratory data (m).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

3. Figure 2
Serum levels of HA in healthy controls and patients with diffuse proliferative LN. (a) HA levels were measured in serum samples obtained from control subjects and from patients with LN during active disease and remission sequentially using a commercial ELISA specific to HA (n=20 for each group). Horizontal lines represent the respective mean value of each group. P<0.001, active LN vs LN in remission or controls. (b) Serum HA levels from active (•) or inactive (○) LN patients correlated with the titers of anti-DNA antibodies.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

4. Figure 3
Comparison of HMC-binding activity between anti-DNA antibodies and serum samples depleted of anti-DNA antibodies (non-anti-DNA immunoglobulins) obtained from lupus patients during inactive and active disease (a). Horizontal line represents the mean value of each group. *P<0.001, Active Ab or inactive Ab vs control IgG. De novo synthesis of 3H-labeled HA by HMC after incubation with control IgG, non-anti-DNA immunoglobulins (non-anti-DNA Ig) or anti-DNA antibodies (10 μg/ml IgG final concentration for all) for 24 h (b). Samples of CM were dialyzed into 8 M urea buffer, pH 6.0, and loaded onto DEAE-Sephacel columns to remove contaminating proteins and glycoproteins. Samples were further purified by Uno Q anion-exchange chromatography and samples eluting with 0.45 M NaCl were collected, digested with papain and subjected to gel filtration chromatography on Sepharose CL-4B under dissociative conditions to separate 3H-labeled HA from digested material. 3H-labeled HA eluted in the void volume of the column and fractions with radioactivity were pooled, and an aliquot subjected to beta scintillation counting to determine total 3H-labeled HA synthesized under control and experiment conditions. Horizontal lines represent the respective mean values for de novo synthesis of 3H-labeled HA in each group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

5. Figure 4
Total 3H-labeled HA synthesized by HMC in relation to the duration of incubation with control IgG (□), Remission Ab (▪) or Active Ab (▪) in the presence of [3H]-glucosamine for periods up to 24 h (a). At select time points, the CM was decanted and the cells extracted with 4 M GuHCl/4% CHAPS. 3H-labeled HA was purified by a combination of dialysis and anion-exchange chromatography. The results are mean±s.d. of three separate experiments. *P<0.05 compared to control IgG. Gel filtration chromatography of 3H-labeled HA synthesized de novo in HMC. Equal aliquots (100 μl) of (b) CM or (c) CE obtained from cells stimulated with control IgG (○), Remission Ab () or Active Ab (•) were subjected to gel filtration chromatography under dissociative condition. The increase in 3H-labeled HA synthesis upon stimulation with Active Ab (upper panels) was abrogated when anti-DNA antibodies were preincubated with exogenous DNA (5 μg/ml) before addition to HMC (bottom panel). P1 and P2 represent HMW and LMW 3H-labeled HA respectively. Vo and Vt represent the void and total volume of the column, respectively.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

6. Figure 5
RT-PCR for HAS II and HAS III mRNA after stimulation with anti-DNA antibodies or control IgG. Total RNA was extracted from HMC using TriReagent according to the manufacturer's instructions and 1 μg of total RNA reverse transcribed to cDNA before PCR amplification for HAS I, II, and III transcripts. (a) PCR products were separated by flat bed electrophoresis on a 2% agarose gel. HAS I was not expressed by HMC. α-Actin was used as the housekeeping gene. (b) Primer to α-actin mRNA densitometric ratios are shown for control IgG (□), Remission Ab (▪) and Active Ab (▪). *P<0.05, control vs Remission Ab; **P<0.001, Active Ab vs control IgG or Remission Ab.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of actinomycin D or cycloheximide on the induction of 3H-labeled HA synthesis in HMC by anti-DNA antibodies. Confluent growth-arrested HMC were incubated in the absence (▪) or presence of actinomycin D (5 μg/ml, ▪) or cycloheximide (5 μg/ml, □) for 1 h before stimulation of cells with control IgG or anti-DNA antibodies for another 24 h. The results show that HA induction by anti-DNA antibodies is dependent on de novo gene transcription and translation. Data represent mean values and s.d. of three individual experiments. *P<0.01, with or without drug treatment, or compared with control IgG.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

8. Figure 7
Effect of anti-DNA antibodies on IL-1β, IL-6, and TNF-α secretion in HMC. Confluent growth-arrested HMC were stimulated with control IgG or anti-DNA antibodies for 24 h. The culture medium was decanted, centrifuged to remove cell debris and the levels of (a) IL-1β, (b) IL-6, and (c) TNF-α determined using commercial ELISAs. Horizontal bars represent the mean value of each group. P<0.001, compared with control IgG or Remission Ab.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

9. Figure 8
(a) Effect of neutralizing antibodies to IL-6 and TNF-α, and IL-1 receptor antagonist on de novo 3H-labeled HA synthesis. HMC were incubated with varying concentrations of neutralizing antibodies to IL-6 and TNF-α, and IL-1 receptor antagonist (0–25 μg/ml) for 1 h before the addition of Active Ab for a further 24 h. The CM was assessed for 3H-labeled HA. *P<0.01, compared to samples not incubated with IL-1 receptor antagonist. (b) Gel filtration chromatography of 3H-labeled HA synthesis. To determine the modulatory effect of IL-1 receptor antagonist on 3H-labeled HA synthesis by Active Ab, HMC were incubated with control IgG in the presence (□) or absence (▪) of IL-1 receptor antagonist (1 μg/ml), or Active Ab in the presence (○) or absence (•) of IL-1 receptor antagonist. Aliquots of CM (upper panel) and CE (lower panel) were chromatographed on Sephacryl S-1000 under dissociative conditions. Vo and Vt represent the void and total volume of the column, respectively.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions