Shigeru Kohno

HSP47 siRNA conjugated with cationized gelatin microspheres suppresses peritoneal fibrosis in mice

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for the biosynthesis and secretion of collagen and is expressed in the fibrotic peritoneum. In the present study, we evaluated the efficacy of HSP47 small interfering RNA (siRNA) to suppress the development of peritoneal fibrosis induced by chlorhexidine gluconate in mice. We initially confirmed that biodegradable cationized gelatin microspheres (CGMs) containing HSP47 siRNA could continuously release siRNA over 21 days as a result of microsphere degradation. We then determined that a single injection of CGMs incorporating HSP47 siRNA suppressed collagen expression and macrophage infiltration, thereby preventing peritoneal fibrosis. Therefore, we suggest that this controlled-release technology using HSP47 siRNA is a potential treatment for peritoneal fibrosis. Additionally, RNA interference combined with CGMs as a drug-delivery system may lead to new strategies for knocking down specific genes in vivo.

Epigallocatechin gallate suppresses peritoneal fibrosis in mice

Long-term peritoneal dialysis (PD) leads to histological changes in the peritoneal membrane. Angiogenesis and inflammation caused by glucose degradation products (GDPs) play crucial roles in peritoneal fibrosis. One such GDP is methylglyoxal (MGO), which enhances the formation of advanced glycation end products (AGEs). AGEs bind to their receptor (RAGE) and activate nuclear factor-κB (NF-κB), which is a key regulator of angiogenesis and inflammation. Recent studies have indicated that (-)-epigallocatechin gallate (EGCG), a tea polyphenol, inhibits angiogenesis and inflammation. Here, we examined whether EGCG suppresses peritoneal fibrosis in mice. Based on preliminary examination, 2mL of 40mM MGO or PD fluid was injected intraperitoneally and EGCG (50mg/kg) or saline was injected subcutaneously for 3weeks. In comparison to PD fluid+saline-treated mice, the peritoneal tissues of MGO+saline-treated mice showed marked thickening of the submesothelial compact zone. In the submesothelial compact zone of the MGO+saline-treated mice, CD31-positive vessels and vascular endothelial growth factor-positive cells were significantly increased, as were inflammation, F4/80-positive macrophages, and monocyte chemotactic protein-1. Moreover, 8-hydroxydeoxyguanosine, a marker of reactive oxygen species, and NF-κB, determined by Southwestern histochemistry, in the submesothelial compact zone were also increased in MGO+saline-treated mice. These changes were attenuated in MGO+EGCG-treated mice. We demonstrated that EGCG treatment suppresses peritoneal fibrosis via inhibition of NF-κB. Furthermore, EGCG inhibits reactive oxygen species production. The results of this study indicate that EGCG is a potentially novel candidate for the treatment of peritoneal fibrosis.

22-Oxacalcitriol Prevents Progression of Peritoneal Fibrosis in a Mouse Model

♦ Objective: Vitamin D plays an important role in calcium homeostasis and is used to treat secondary hyperparathyroidism among dialysis patients. The biologic activity of vitamin D and its analogs is mediated by vitamin D receptor (VDR), which is distributed widely throughout the body. Recent papers have revealed that low vitamin D levels are correlated with severe fibrosis in chronic diseases, including cystic fibrosis and hepatitis. The aim of the present study was to evaluate the protective effects of vitamin D against the progression of peritoneal fibrosis. ♦ Methods: Peritoneal fibrosis was induced by injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. An analog of vitamin D, 22-oxacalcitriol (OCT), was administered subcutaneously daily from initiation of the CG injections. The peritoneal tissue was excised at 3 weeks. Changes in morphology were assessed by hematoxylin and eosin staining. Expression of VDR, alpha smooth muscle actin (as a marker of myofibroblasts), type III collagen, transforming growth factor β(TGF-β), phosphorylated Smad2/3, F4/80 (as a marker of macrophages), and monocyte chemoattractant protein-1 (MCP-1) was examined by immunohistochemistry. Southwestern histochemistry was used to detect activated nuclear factor κB (NF-κB). ♦ Results: In the CG-injected mice, immunohistochemical analysis revealed expression of VDR in mesothelial cells, myofibroblasts, and macrophages in the thickened submesothelial zone. Treatment with OCT significantly prevented peritoneal fibrosis and reduced the accumulation of type III collagen in CG-treated mice. Among the markers of fibrosis, the numbers of myofibroblasts, cells positive for TGF-β, and cells positive for phosphorylated Smad2/3 were significantly decreased in the OCT-treated group compared with the vehicle-treated group. Furthermore, OCT suppressed inflammatory mediators of fibrosis, as shown by the reduced numbers of activated NF-κB cells, macrophages, and MCP-1-expressing cells. ♦ Conclusions: Our results indicate that OCT attenuates peritoneal fibrosis, an effect accompanied by reduced numbers of myofibroblasts, infiltrating macrophages, and TGF-β-positive cells, suggesting that vitamin D has potential as a novel therapeutic agent for preventing peritoneal sclerosis.

Recombinant human erythropoietin attenuates renal tubulointerstitial injury in murine adriamycin-induced nephropathy

BACKGROUNDnErythropoietin (EPO) has been found to provide cytoprotection against acute ischemic and toxic renal tubulointerstitial injury. This study aimed to elucidate the mechanism(s) underlying EPO protection while examining whether EPO provides tubulointerstitial protection in a mouse model with adriamycin (ADR)-induced tubulointerstitial injury.nnnMETHODSnAdriamycin nephropathy (AN) was induced by a single injection of ADR in the 2 experimental groups on day 0. The saline-control group and the AN-saline group were administered saline at days 7, 14, and 21, while the EPO-control group and the AN-EPO group were administered EPO at days 7, 14, and 21. Kidneys were harvested at days 14 and 28 after ADR injection to measure the expression levels of the EPO receptor (EPO-R), CD34, and phosphorylated Akt by immunohistochemistry; to determine the extent of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 staining; and to map the hypoxic area by pimonidazole staining.nnnRESULTSnEPO-R was detected in glomerular, tubular epithelial, and endothelial cells. EPO administration significantly improved tubulointerstitial injury, decreased the number of TUNEL-positive and active caspase-3-positive cells, and increased the phosphorylated-Akt-positive area in the tubulointerstitial area without increasing the hemoglobin or hematocrit levels.nnnCONCLUSIONSnEPO provides renoprotection against AN by reducing apoptotic cell death and preserving peritubular capillaries, possibly by exerting pleiotropic effects independently of its hemopoietic effects.

SAHA Suppresses Peritoneal Fibrosis in Mice.

♦ Objective: Long-term peritoneal dialysis causes peritoneal fibrosis in submesothelial areas. However, the mechanism of peritoneal fibrosis is unclear. Epigenetics is the mechanism to induce heritable changes without any changes in DNA sequences. Among epigenetic modifications, histone acetylation leads to the transcriptional activation of genes. Recent studies indicate that histone acetylation is involved in the progression of fibrosis. Therefore, we examined the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on the progression of peritoneal fibrosis in mice. ♦ Methods: Peritoneal fibrosis was induced by the injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. SAHA, or a dimethylsulfoxide and saline vehicle, was administered subcutaneously every day from the start of the CG injections for 3 weeks. Morphologic peritoneal changes were assessed by Masson’s trichrome staining, and fibrosis-associated factors were assessed by immunohistochemistry. ♦ Results: In CG-injected mice, a marked thickening of the submesothelial compact zone was observed. In contrast, the administration of SAHA suppressed the progression of submesothelial thickening and type III collagen accumulation in CG-injected mice. The numbers of fibroblast-specific protein-1-positive cells and α-smooth muscle actin α-positive cells were significantly decreased in the CG + SAHA group compared to that of the CG group. The level of histone acetylation was reduced in the peritoneum of the CG group, whereas it was increased in the CG + SAHA group. ♦ Conclusions: Our results indicate that SAHA can suppress peritoneal thickening and fibrosis in mice through up-regulation of histone acetylation. These results suggest that SAHA may have therapeutic potential for treating peritoneal fibrosis.

Involvement of Leptin in the Progression of Experimentally Induced Peritoneal Fibrosis in Mice

Leptin is a hormone mainly produced by white adipose cells, and regulates body fat and food intake by acting on hypothalamus. Leptin receptor is expressed not only in the hypothalamus but in a variety of peripheral tissues, suggesting that leptin has pleiotropic functions. In this study, we investigated the effect of leptin on the progression of peritoneal fibrosis induced by intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 2 or 3 weeks in mice. This study was conducted in male C57BL/6 mice and leptin-deficient ob/ob mice. Peritoneal fluid, blood, and peritoneal tissues were collected 15 or 22 days after CG injection. CG injection increased the level of leptin in serum and peritoneal fluid with thickening of submesothelial compact zone in wild type mice, but CG-injected ob/ob mice attenuate peritoneal fibrosis, and markedly reduced the number of myofibroblasts, infiltrating macrophages, and blood vessels in the thickened submesothelial area. The 2-week leptin administration induced a more thickened peritoneum in the CG-injected C57BL/6 mice than in the PBS group. Our results indicate that an upregulation of leptin appears to play a role in fibrosis and inflammation during peritoneal injury, and reducing leptin may be a therapeutically potential for peritoneal fibrosis.

Involvement of Lymphocyte Infiltration in the Progression of Mouse Peritoneal Fibrosis Model

Peritoneal fibrosis is a serious complication in patients with severe chronic kidney disease who are undergoing peritoneal dialysis (PD). One of the pathological characteristics of peritoneal fibrosis is the infiltration of macrophages in the thickened submesothelial compact zone. In addition, infiltration of lymphocytes, including T and B lymphocytes, is observed in the fibrotic peritoneum. However, the relationship between lymphocyte infiltration and progression of peritoneal fibrosis remains unclear. In this study, we investigated the role of lymphocytes in the development of peritoneal fibrosis induced by chlorhexidine gluconate (CG) by comparing the histological changes observed in severe combined immunodeficient (SCID) mice (largely lacking functional T and B lymphocytes) with those observed in wild-type (WT) mice. As expected, CG-injected WT mice showed a thickening of the submesothelial compact zone together with massive collagen deposition accompanied by increased numbers of infiltrating macrophages and T and B lymphocytes. In the peritoneum of SCID mice, the submesothelial compact zone was thicker and the number of macrophages and B lymphocytes was significantly higher than that observed in control immunodeficient and WT mice. In contrast, the number of T lymphocytes in the peritoneum of SCID mice was significantly lower than that in the peritoneum of WT mice. These results suggest that T and B lymphocytes modulate the process of peritoneal fibrosis via macrophage infiltration.

New Insights into Therapeutic Strategies for the Treatment of Peritoneal Fibrosis: Learning from Histochemical Analyses of Animal Models

Encapsulating peritoneal sclerosis (EPS) is a fatal complication that can occur in patients undergoing long-term peritoneal dialysis. It is characterized by bowel obstruction and marked sclerotic thickening of the peritoneal membrane. Although the mechanisms underlying the development of EPS are complex, angiogenesis, inflammation, and peritoneal fibrosis are known to be essential factors. Now, several animal models that exhibit EPS have pathophysiology similar to that of human EPS and have been proposed for use in research to provide insights into it. Recent histochemical methods also help us to understand the pathophysiology of EPS. Advances in basic research based on the findings in those animal models have enabled the development of several strategies for the prevention and treatment of EPS. We describe here interventional studies in some animal models for peritoneal fibrosis, one of the histological disorders findings characteristic to EPS, and we highlight the need for a sophisticated animal model that closely resembles human conditions.

Production and degradation of extracellular matrix in reversible glomerular lesions in rat model of habu snake venom-induced glomerulonephritis.

We investigated the mechanism of development and repair process of glomerular injury in a rat model of habu snake (Trimeresurus flavoviridis) venom (HSV)-induced glomerulonephritis. Glomerulonephritis was induced in rats by intravenously injecting HSV at 3 mg/kg. Renal tissue was isolated and subjected to immunohistochemical analysis for expression levels of type IV collagen, heat shock protein 47 (HSP47), transforming growth factor-β (TGF-β), and matrix metalloproteinase-3 (MMP-3), as well as its transcription factor Ets-1. Expression levels of HSP47, TGF-β, and type IV collagen began to increase in the mesangial area starting from day 14 and peaked on day 21, followed by a gradual decrease. Expression levels of MMP-3 and Ets-1 started to increase coinciding with peak production of mesangial matrix on day 21, peaking on day 35, followed by gradual decrease. Expression of MMP-3 and Ets-1 persisted until day 63, whereas that of HSP47 and type IV collagen returned to baseline level at this time point. Time-course changes of extracellular matrix (ECM) accumulation in glomeruli in the HSV-induced glomerulonephritis model were correlated with those of factors involved in both ECM production and degradation systems. Continued expression of factors in the degradation system seems particularly important for the repair process. These findings might lead to new therapies that prevent and repair glomerular injury.