Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells.

UNLABELLED: Emodin ameliorates glucose-induced matrix synthesis in human peritoneal mesothelial cells. Prolonged exposure of human peritoneal mesothelial cells (HPMC) to high glucose concentrations in peritoneal dialysate is the principal factor leading to matrix accumulation and thickening of the peritoneal membrane, accompanied by progressive deterioration of transport functions. These changes are mediated in part through protein kinase C (PKC) activation and the induction of transforming growth factor-beta 1 (TGF-beta 1). Emodin (3-methyl-1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells. How emodin modulates glucose-induced abnormalities in HPMC has not been elucidated and thus constitutes the theme of this study. METHODS: We investigated the effects of emodin on the expression of PKC alpha, TGF-beta 1, fibronectin, and collagen type I in HPMC, and its effects on HPMC proliferation under physiologic (5 mmol) or high (30 mmol) glucose concentrations. RESULTS: Exposure of HPMC cultured with 5 mmol or 30 mmol D-glucose to emodin (20 microg/mL) resulted in an initial lag of proliferation by 2.3 to 2.7 days, but did not affect cell viability or morphology at confluence. D-glucose (30 mmol) induced TGF-beta 1 secretion in a time-dependent manner (3.72 +/- 0.29 and 4.30 +/- 0.50 pg/microg cellular protein at 24 hours and 48 hours respectively, compared to 2.13 +/- 0.23 and 2.65 +/- 0.32 pg/microg cellular protein at 24 hours and 48 hours, respectively for 5 mmol glucose; P < 0.001 at both time points). Such induction was ameliorated by emodin (20 microg/mL) (TGF-beta 1 concentration 2.25 +/- 0.15 and 2.96 +/- 0.33 pg/microg cellular protein at 24 hours and 48 hours, respectively, in the presence of emodin and 30 mmol D-glucose; P < 0.001 compared to 30 mmol D-glucose alone at both time points). Induction of TGF-beta 1 synthesis by 30 mmol D-glucose was associated with induction of PKC alpha, phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor-1 (ATF-1), and increased fibronectin and type I collagen translation. Emodin abrogated all these effects of concentrated glucose. Immunohistochemical staining showed that 30 mmol D-glucose induced cytoplasmic, perinuclear, and extracellular fibronectin and type I collagen expression by HPMC. Emodin reduced 30 mmol D-glucose-induced cytoplasmic and extracellular matrix synthesis to near basal levels. CONCLUSION: Our findings demonstrate that emodin ameliorates the undesirable effects of concentrated glucose on HPMC via suppression of PKC activation and CREB phosphorylation, and suggest that emodin may have a therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities in the peritoneal membrane.

Different effects of amino acid-based and glucose-based dialysate from peritoneal dialysis patients on mesothelial cell ultrastructure and function.

BACKGROUND: Peritoneal dialysis fluid (PDF) containing amino acids has been introduced recently aiming to improve the nutritional status of PD patients. Dextrose-based PDFs have been implicated in progressive functional and structural deterioration of the peritoneal membrane. Limited data are currently available regarding the effect of amino acid-based PDF on the function and ultrastructure of human peritoneal mesothelial cells (HPMCs), which play a critical role in peritoneal membrane pathophysiology. METHODS: We investigated the effects of two commercially available PDFs, which utilized dextrose (1.5% Dianeal) or amino acids (1.1% Nutrineal) as the osmotic agent, obtained from patients after a 4 h dwell, on HPMC proliferation (MTT assay and cell counting) and viability [lactate dehydrogenase (LDH)release], interleukin-6 (IL-6) secretion (commercial enzyme-linked immunosorbent assay) and ultrastructure (scanning and transmission electron microscopy). RESULTS: Exposure of HPMCs to 1.5% Dianeal reduced cell proliferation, total cellular protein synthesis, IL-6 secretion and cell attachment, but prolonged the cell doubling time on recovery, and increased LDH release (P<0.001, P<0.001, P<0.0001, P<0.0001, P<0.001 and P<0.001, respectively). The 1.1% Nutrineal reduced HPMC proliferation (P<0.001) and increased IL-6 secretion (P<0.0001), but did not affect cell attachment, LDH release, protein synthesis or cell doubling time. Ultrastructural studies of HPMCs exposed to Dianeal showed cell flattening, increased cell surface area, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after incubation with Nutrineal. CONCLUSIONS: Our results showed that HPMC ultrastructure, viability and protein synthesis were better preserved with amino acid-based PDF, compared with conventional dextrose-based PDF. The significance of IL-6 induction by Nutrineal remains to be elucidated.

Mesangial cell-binding anti-DNA antibodies in patients with systemic lupus erythematosus.

The mechanisms by which anti-DNA antibodies contribute to the pathogenesis of lupus nephritis (LN) remain to be elucidated. This study investigates the binding of polyclonal anti-DNA immunoglobulins from patients with systemic lupus erythematosus (SLE) to human mesangial cells (HMC) in vitro. Testing of cross-sectional serum samples from 280 LN patients (108 during active disease; 172 during remission), 35 SLE patients without renal involvement, 72 patients with non-lupus primary glomerular diseases, and 37 healthy subjects with a cellular enzyme-linked immunosorbent assay showed significant IgG mesangial cell-binding activity in patients with SLE, particularly those with active LN (P < 0.0001). Significant HMC-binding activity was demonstrated in 83.9%, 42.8%, and 47.1% of patients with active LN, inactive LN, and non-renal SLE, respectively. This was predominantly attributed to binding by anti-DNA antibodies, and immune complex binding accounted for 4.6%, 3.5%, and 2.8% of seropositive samples in the respective groups. Longitudinal studies in 27 LN patients demonstrated correlation between serial levels of anti-DNA antibodies, serum HMC-binding activity, and disease activity in 18 patients (66.7%). Affinity-purified polyclonal IgG anti-DNA antibodies from sera with HMC-binding activity showed significant binding to cultured HMC, and to a lesser extent glomerular and proximal tubular epithelial cells and human umbilical vein endothelial cells, but not tumor cell lines, peritoneal mesothelial cells, bronchial epithelial cells, or fibroblasts. The binding of anti-DNA antibodies to HMC was increased 1.47-fold (P = 0.0059) after the removal of Ig-associated DNA by DNase treatment, but it was unaffected by DNase treatment of HMC membrane. Controlled trypsinization of membrane proteins in HMC resulted in a 1.26-fold (P = 0.0025) increase in their binding by anti-DNA antibodies. In conclusion, subsets of anti-DNA antibodies from patients with SLE are capable of binding to HMC. The association of such binding with renal involvement and disease activity and its modulation by DNA concentration suggest that Ig binding to HMC can be a potential marker for disease activity in selected patients and that the binding of anti-DNA antibodies to HMC may be a pathogenetic mechanism in LN.